Nucleic acid complexes

ABSTRACT

The present invention relates to complexes of transcription factor decoys, their delivery to bacteria and their formulation. In particular, the present invention resides in an antibacterial complex comprising a nucleic acid sequence and one or more delivery moieties selected from quaternary amine compounds; bis-aminoalkanes and unsaturated derivatives thereof, wherein the amino component of the aminoalkane is an amino group forming part of a heterocyclic ring; and an antibacterial peptide.

The present invention relates to complexes of nucleic acid sequences. Inparticular, the present invention relates to complexes of transcriptionfactor decoys, their delivery to bacteria and their formulation.

Control of bacterial growth and virulence poses an increasing problem,particularly in medical and veterinary applications, and may become amajor challenge to public health. Antibiotics for use against pathogenicbacteria are well known in the art. However, extensive use of suchantibiotics has led to the emergence of bacteria which are resistant toat least one, and in some cases, multiple antibiotics (so-calledmulti-drug resistant strains). The situation is exacerbated by adecrease in the numbers of conventional antibiotics being discovered andunder development. Indeed, antibiotic resistance is a major challengefor antibacterial research and threatens the potency of marketedantibiotics as well as those still under development. Consequently thereis a need for new anti-bacterial agents which can be used to tacklebacterial spread and infection.

DNA-based therapies have the potential to overcome the limitations ofexisting antibacterial therapies because they can be designed to treatpotentially any pathogen either by preventing expression of genesencoding an antibiotic resistance mechanism or by inhibiting growth bymodifying the expression of essential or adaptive genes or operons orsimilarly preventing onset of virulence or pathogenicity. In addition,bacteria are unlikely to develop resistance to these agents as thiswould require simultaneous mutations that affected both thetranscription factor and its cognate binding site(s). This isparticularly true for agents that control several essential genes.Alternatively a gene could evade transcription factor decoy-mediatedcontrol by mutation so that the gene is expressed constitutively.However, in the case of a transcription factor decoy that actssimultaneously on many genes, it would require many of those genes toacquire mutations to switch them to constitutive expression, an eventthat is considered unlikely.

Transcription Factor Decoys (TFDs) are one such DNA-based therapeutic.Decoy oligonucleotides are designed to mimic the binding sites oftranscription factors and prevent the latter from binding to theircognate genomic targets, with a consequent modification of geneexpression (Mann and Dzau (2000) J. Clin. Investigation 106: 1071-1075).

TFDs have distinct advantages over other DNA-based therapeutics. Theirmechanism of action is simple and predictable—they control geneexpression by sequestering transcription factors, preventing the latterfrom binding to promoters by flooding the cell with sufficient copies ofthe specific binding sequences (hence, the term “decoys”). This is incontrast to antisense strategies where targets are difficult to definedue to the complex secondary structure of mRNA. In comparison toantisense approaches, TFDs have the further advantages that they actrapidly, preventing expression of genes, whereas antisense approachesdeal with the consequences of expression. As a result, TFDs areeffective at much lower concentrations, because a singleTFD-transcription factor interaction can block transcription of a singlegene that otherwise may have given rise to many thousands of copies ofmRNA, which constitute the targets for the antisense approach.

Other DNA-based therapeutics include plasmids containing transgenes forgene therapy, oligonucleotides for antisense and antigene applications(Crooke S. T. (1998) Antisense Nucleic Acid Drug Dev. 8: 115-122),ribozymes, DNAzymes, aptamers, and small interfering RNAs (siRNAs)(Stull R. A. et al (1995) Pharm Res. 12: 465-483; Patil S. D. andBurgess D. J. (2003) AAPS Newsmagazine 6: 27). Although most of theDNA-based drugs are in pre-clinical development or the early stages ofclinical trials, this class of compounds has emerged in recent years toyield extremely promising candidates for drug therapy for a wide rangeof diseases, including cancer, AIDS, neurological disorders such asParkinson's disease and Alzheimer's disease, and cardiovasculardisorders.

However, delivery of DNA-based therapeutics, including TFDs, remains asignificant challenge. Cell membranes are one of the major obstaclesencountered when delivering large hydrophilic therapeutic agents such asproteins or nucleic acids. Most effort to solve this problem is focusedon delivery to eukaryotic cells, to develop effective therapies forhuman cancer and the like. In eukaryotes the different cellularcompartments (including the nucleus) are protected by biologicalmembranes which segregate the various cellular compartments and preventinflux and efflux of solutes from cells and organelles. Although thesebarriers are essential for the maintenance of the cell, they are asubstantial problem when trying to deliver therapeutics to a cell orspecific organelles within a cell.

Most cations are unable to pass through cell membranes without specificcarrier systems due to the large free energy barrier posed by thehydrophobic interior of the membrane. To be selectively accumulated andretained by a cell, a cationic compound requires sufficientlipophilicity and delocalisation of its positive charges to reduce thefree energy change when moving from an aqueous to a hydrophobicenvironment. This allows the compound to cross the plasma andmitochondrial membranes passively and be accumulated in response tomembrane potential.

Transport of proteins into eukaryotic cells is mainly mediated throughendocytosis. This is a highly organised transport system in which largepolar molecules are absorbed by being engulfed in the cell membrane.Phagocytosis and pinocytosis are non-receptor-mediated forms ofendocytosis while receptor-mediated endocytosis is a more specificactive event where the cytoplasm membrane folds inward to form coatedpits. In this case, proteins or other trigger particles lock intoreceptors/ligands in the cell's plasma membrane or by non-specificinteraction with the surface of the cell, for example due to chargeinteractions. It is only then that the particles are engulfed. Theseinward budding vesicles bud to form cytoplasmic vesicles.

Various techniques have been developed to translocate biologicallyactive molecules across these barriers in vivo and in vitro.Electroporation and microinjection are harsh and impractical to use invivo because these methods necessitate disruption of the cell membranebefore substances can be introduced into the cell and therefore they arerestricted to a limited amount of cells.

While use of the endocytotic pathway is appealing, one significantdrawback is ensuring endosomal escape before lysosomal activity occurs.Liposome encapsulation and receptor-mediated endocytosis are alsolimited by their lack of targeting and low yield of delivery. Most ofthe exploratory work carried out to date has focussed on the delivery ofsiRNA molecules to eukaryotic cells. The approach usually taken is toform liposomes or complexes with covalently attached targeting peptideswhich direct the therapeutic to the appropriate target within the cell.

There are many considerations when selecting the lipid component forsuch a delivery mechanism. The lipid-like component is generallycationic and has three functions:

-   -   1. to condense the nucleic acid to make delivery more        achievable;    -   2. to protect the nucleic acid from degradation by nucleases or        non-specific absorption by plasma proteins etc., and    -   3. to shield the negative charge of phosphate backbone.

The third point can be overcome by using synthetic backbones withreduced or no charge.

In addition, liposomes or complexes require certain ideal physicalproperties:

-   -   1. Good stability, both in salt or biological fluids. This can        be quantified with the ξ-potential which measures the attraction        or repulsion between the particles;    -   2. Narrow size distribution, for bacteria ideally between 50 and        100 nm;    -   3. Lower toxicity, although most transfection agents (lipids,        lipophilic cations) show moderate levels of toxicity;    -   4. Practical to produce, incorporating issues such as cost of        goods.

Peptides used for eukaryotic delivery are generally known topermeabilise membranes, to be cell-penetrating via specific receptors ontarget cells, or simply to be cationic (e.g. poly-arginine). The use ofpeptides with cell-penetrating properties has several advantages, whichare mainly due to the various modifications that can be made to thepeptide sequence. This allows the engineering of carriers addressingdifferent cellular subdomains and/or able to transport various types ofcargoes.

A class of membrane translocating agents is the cell-penetratingpeptides (CPPs). Apart from being a mild and effective tool for accessto different cellular organelles in vitro, CPPs have been used forcellular delivery of several agents in vivo, with promising results.

CPPs generally consist of less than 30 amino acids, have a net positivecharge and have the ability to translocate the plasma membrane andtransport several different cargoes into the cytoplasm and nucleus in aseemingly energy-independent manner. Translocation occurs via an as yetunknown mechanism that is not affected by various endocytosis inhibitorsor low temperatures (Langel, Ü., ed. (2002) Cell-Penetrating Peptides,Processes and Applications, CRC Press).

The term CPP includes synthetic cell-permeable peptides,protein-transduction domains and membrane-translocating sequences, whichall have the ability to translocate the cell membrane and gain access tothe cellular interior.

Other classes of peptides with the potential to affect deliveryespecially to bacteria are those with antibacterial properties. Theseinclude the Anti-Microbial Peptides (AMPs), cell penetrating peptides(CPPs), those peptides that are synthesised non-ribosomally andglycopeptides. All these types of peptides are referred to herein asAnti-Bacterial Proteins (ABPs).

AMPs can be derived from bacterial sources, where they are referred toas bacteriocins, or from higher eukaryotic sources, including humans,amphibians, insects etc. AMPs of particular interest are those that areable to either increase the permeability of biological membranes or havethe ability to translocate across membranes to reach their intracellulartargets. AMPs are generally divided into five non-mutually exclusivesub-groups on the basis of structural similarities (Brogden, 2005 Nat.Rev. Microbiol. 3: 238-250):

-   -   1. Anionic peptides are small peptides commonly complexed with        zinc and active against Gram-negative and Gram-positive        bacteria;    -   2. Linear cationic α-helical peptides (such as buforin) that are        less than 40 amino acids in length, lack cysteines and feature a        central hinge region between two alpha-helical regions;    -   3. Cationic peptides that are enriched for specific amino acids,        such as proline- and arginine-rich apidaecins;    -   4. Anionic and cationic peptides that form beta-sheets due to        formation of disulphide bonds between cysteine groups;    -   5. Anionic and cationic peptides that are fragments of larger        proteins.

The mechanisms by which the AMPs affect microbial killing are varied andnot confined to certain sub-groups. The majority kill bacteria byforming pores in their membranes leading to the cells beingpermeabilised. There are thought to be three distinct mechanisms ofpermeabilisation, which can either be a specific property of a peptideor an effect of the concentration of the peptide used:

-   -   1. Formation of torroidal pores by, for example, magainin,        melittin;    -   2. Carpet formation by, for example, dermaseptin S, cecropin or        melittin;    -   3. Barrel stave formation by, for example, alamethicin.

Several AMPs have been described where the final target is not themembrane but intracellular in nature, necessitating that the AMPtranslocates across the membrane. This ability to translocate is ofparticular interest as these peptides, or synthetic peptides modelled onthem, could be used to deliver alternative cargoes such as therapeutics.These AMPs can therefore be classed according to their modes ofintracellular killing:

-   -   1. Binding of nucleic acids by, for example, buforin, buforin II        and analogues and tachyplesin;    -   2. Flocculation of intracellular contents by, for example,        anionic peptides;    -   3. Septum formation by, for example, microcin 25;    -   4. Cell wall synthesis by, for example, mersacidin;    -   5. Inhibition of nucleic-acid synthesis by, for example,        pleurocidin and dermaseptin;    -   6. Inhibition of protein synthesis by, for example, pleurocidin        and dermaseptin;    -   7. Inhibition of enzymatic activity by, for example, apidaecins.

Non-ribosomally synthesised peptides can be structurally diverse. Theyare often cyclised or branched, contain non-standard amino acids oramino acids that have been modified to produce hydroxyalanine orhydroxyserine, and can be subject to hydroxylation, halogenation orglycosylation (Walsh et al (2004) Science 303: 1805-1810). The majorityof these peptides inhibit growth of bacteria by permeabilising theirmembranes.

The challenges of peptide and nucleic acid delivery to bacteria aresimilar to those encountered with eukaryotic cells but the cellularcomponents are different. In particular, bacteria do not have anendocytic mechanism to help moving the therapeutic into the cell.However, a clear advantage is that bacteria do not have a nuclearcompartment so it is sufficient to deliver the therapeutic into thecytoplasm for it to gain access to the bacterial genome. Therefore,alternatives to eukaryotic delivery systems need to be developed. Theseinvolve finding ways of gaining entry to the cell by permeabilising themembrane(s). In a laboratory setting, delivery of DNA into cells(transfection) is achieved by either treating the cells with anionicbuffers that disrupt the cell membranes by interfering with chargedistribution, most commonly by buffers containing calcium ions.Alternatively electroporation is used, where cells are prepared inbuffers with low conductance and high voltages are used to producetransient pores in the bacterial membranes of some of the cells, thoughby no means all, resulting in transfection of a minority of the cells.

It is evidently impractical to use either method to deliver DNAtherapeutics to bacteria either in animal models or in a clinicalsetting. Transfection in vivo may be achieved by conjugating thetherapeutic to a synthetic cationic peptide or ABP known to damage themembranes and cause entry through the subsequent pores or extrusions.Additionally the ability of some of the ABPs to translocate throughmembranes is another alternative in developing a credible deliverystrategy.

Starting from the eukaryotic model of using delivery peptides, idealfeatures for bacterial delivery peptides include:

-   -   1. bacterial-specificity/preference, being optimised for        prokaryotes rather than eukaryotes;    -   2. low likelihood of eliciting resistance. Cationic peptides,        for example, can be resisted by MRSA which alters the charge        density of its outer membrane;    -   3. broad-spectrum activity so for use as a general delivery        system to bacteria; and    -   4. low or no host toxicity.

Similarly, the use of lipid-like transport molecules needs to betailored to the specific construction of bacterial membrane. Forexample, Gram-negative bacteria have an outer and an inner membrane,both of which need to be overcome before the therapeutic is deliveredinto the bacterium.

Against this background, the present invention provides a solution forthe formulation and delivery of nucleic acid sequences, such as TFDs, tobacteria.

Specifically, the present invention resides in an antibacterial complexcomprising a nucleic acid sequence and one or more delivery moieties.The inventors have found that delivery moieties that have someantibacterial activity in their own right are ideal. In particular, thedelivery moiety or moieties should show a synergistic antibacterialeffect when combined with the nucleic acid sequence. In other words, thecombination of the delivery moiety and the nucleic acid sequence shouldshow an enhanced antibacterial effect when compared to the effect of thenucleic acid sequence or delivery moiety alone.

The delivery moiety may be selected from quaternary amine compounds andbis-aminoalkanes and unsaturated derivatives thereof, wherein the term“aminoalkanes” as used herein refers to amino groups (preferablytertiary amino groups) that form part of a heterocyclic ring.

Exemplary of such compounds are compounds of the formula (I):Q-(CH₂)_(p)-A-(CH₂)_(q)—R³  (I)wherein Q is selected from:

-   (a) a group Q¹ having the formula:

and

-   (b) a group Q², Q²-NH—, Q²-O—, or Q²-S— wherein Q² is selected from    monocyclic, bicyclic and tricyclic heteroaromatic groups of 5 to 14    ring members, of which 1, 2 or 3 are heteroatom ring members    selected from N, O and S provided that at least one nitrogen ring    member is present, wherein the heteroaromatic groups are optionally    substituted by one or two substituents R^(4a) and wherein the said    one nitrogen ring member may form an N-oxide or may be substituted    with C₁₋₄ alkyl, phenyl-C₁₋₄ alkyl or di-phenyl-C₁₋₄ alkyl to form a    quaternary group, wherein the phenyl moieties in each case are    optionally substituted with one or two halogen, methyl or methoxy    groups;-   m is 0 or 1;-   n is 0 or 1;-   p and q are the same or different and each is an integer from 1 to    12;-   A is a bond or is selected from a naphthalene, biphenyl, terphenyl,    phenanthrene, fluorene, stilbene, a group C₆H₄(CH₂)_(r)C₆H₄, a group    C₆H₄—C≡C—C₆H₄, a pyridine-2,6-diyl-bis(benzene-1,4-diyl) group, a    group CH═CH—(CH₂)_(s)—(CH═CH)_(t)—; and a group    C≡C—(CH₂)_(u)—(C≡C)_(v)—; wherein r is 0-4, s is 0 to 4, t is 0 or    1; u is 0-4 and v is 0 or 1;-   when n is 1, R⁰, R¹ and R² are each selected from C₁₋₄ alkyl; and    when n is 0, then N, R¹ and R² together form a monocyclic, bicyclic    or tricyclic heteroaromatic group of 5 to 14 ring members, of which    one is the nitrogen atom N and 0, 1 or 2 are further heteroatom ring    members selected from N, O and S, and wherein the heteroaromatic    group is optionally substituted by one or two substituents R^(4b);    and-   R³ is selected from hydrogen, C₁₋₄ alkyl, halogen, monocyclic    carbocyclic groups of 3 to 7 ring members each optionally    substituted by one or two substituents R^(4c), a group Q; a group    —NH-Q², a group —O-Q² and a group —S-Q²; and-   R^(4a), R^(4b) and R^(4c) are the same or different and each is    selected from C₁₋₄ alkyl optionally substituted with one or more    fluorine atoms: C₁₋₄ alkoxy optionally substituted with one or more    fluorine atoms; nitro; amino; mono- and di-C₁₋₄ alkylamino; halogen;    phenyl-C₁₋₂ alkyl wherein the phenyl moiety is optionally    substituted with one or two methoxy, methyl or halogen substituents;    ureido and guanidinyl.

In one preferred embodiment, Q is a group Q¹:

Accordingly, one preferred sub-group of compounds within formula (I) isrepresented by formula (II):

wherein R¹, R², m, p, A, q and R³ are as defined in respect of formula(I).

One preferred sub-group of compounds within formula (II), wherein R³ isQ¹ and n in each instance is 0, can be represented by the formula (III):

wherein R¹, R², m, p, A and q are as defined in respect of formula (I).

In the compounds of formulae (I), (II) and (III), when m is 1, thenitrogen atom N must be a quaternary nitrogen. Accordingly, thecompounds of formulae (I), (II) and (Ill) wherein m is 1 will compriseone or more anions as counter ions, for example anions derived frommineral acids, sulphonic acids and carboxylic acids.

When N, R¹ and R² together form a monocyclic, bicyclic or tricyclicheteroaromatic group of 5 to 14 ring members, typically the groupcontains either the nitrogen atom N as the sole heteroatom ring memberor contains a second heteroatom ring member selected from N, O and S.

When N, R¹ and R² together form a monocyclic, bicyclic or tricyclicheteroaromatic group, the nitrogen atom N forms part of an aromaticring. Preferred heteroaromatic groups are monocyclic aromatic rings;bicyclic heterocyclic rings in which both rings are aromatic; bicyclicheterocyclic rings in which one nitrogen-containing ring is aromatic andthe other ring is non-aromatic; and tricyclic rings in which two rings,including a nitrogen-containing ring, are aromatic and the other ring isnon-aromatic.

When N, R¹ and R² together form a monocyclic, bicyclic or tricyclicheterocyclic group of 5 to 14 ring members, the heterocyclic group ispreferably selected from quinoline; isoquinoline; acridine;tetrahydroacridine and ring homologues thereof; pyridine;benzoimidazole; benzoxazole and benzothiazole. By ring homologues oftetrahydroacridine is meant compounds containing the core structure:

wherein y is 1 or 3. By tetrahydroacridine is meant a compound havingthe core structure above wherein y is 2.

In a preferred embodiment, the delivery moiety is a quaternaryderivative of quinoline or acridine, in particular 1, 2, 3,4-tetra-hydro-9-amino-acridine. Suitable derivatives of 1, 2, 3,4-tetra-hydro-9-amino-acridine are described in U.S. Pat. No. 3,519,631,the contents of which are incorporated herein by reference. Ofparticular interest are the compounds and formulae exemplified inExamples 17 to 25 of U.S. Pat. No. 3,519,631 and analogues thereof.Suitable quinoline derivatives are the bis-quinolinium compounds, suchas dequalinium, and analogues thereof.

Dequalinium (FIG. 1) is a bis-quinolinium compound, has mildantimicrobial properties and has been used for 30 years as anantimicrobial agent in over-the-counter mouthwashes, topical ointments,oral and vaginal paints, and sore-throat lozenges. It is a topicalbacteriostat and has also been tested as an inhibitor of calciumchannels, an antifungal agent (Ng et al. (2007) Bioorg. Medicinal Chem.15: 3422-3429), an inhibitor of Tuberculosis (Guiterrez-Lugo et al.(2009) J. Biomol. Screen. 14: 643-652) and as an inhibitor of ProteinKinase C (PKC; Abeywickrama et al. (2006) Bioorg. Medicinal Chem. 14:7796-7803).

Dequalinium has also been used to deliver DNA-based therapeutics andconventional drugs, such as paxcitol, to mitochondria in in vitroexperiments. In these experiments, dequalinium was prepared as bolasomesby the dry-film method. That is, dequalinium is dissolved in an organicsolvent, such as methanol, dried to completion in a vacuum andre-suspended in an aqueous solution, whereupon it is sonicated to formbolasomes which are subsequently mixed with DNA to form complexes. Thesecomplexes have been shown to be capable of delivering DNA tomitochondria by a mechanism which is believed to involve fusion of thecomplexes with the outer membrane of the mitochondria (Weissig andTorchilin 2001 Adv. Drug Delivery Rev. 49: 127-149; Weissig et al. 2001J. Control. Release 75: 401-408). Although the membrane structure ofmitochondria is not equivalent to those that occur in bacteria, it issimilar. This raises the possibility that dequalinium could be suitableto deliver therapeutics to bacteria in an in vivo setting. However, thecomplexes formed with dequalinium are unstable over time in the presenceof physiological buffers and biological fluids and to dilution.

Therefore, in a particularly preferred embodiment, the complex comprisesa dequalinium analogue. In this way, it is possible to design adequalinium compound that has enhanced stability (both to dilution andthe presence of salt) and yet has a similar or improved toxicityprofiles to dequalinium. Such an analogue has been described that formsmore stable complexes (Compound 7, Galanakis et al. [J. Med. Chem.(1995) 35: 3536-3546]) as tested by various physiochemical parameterssuch as ability to bind DNA to the exclusion of fluorescent dyeSYBR-green, size of particles formed as measured by Dynamic LightScattering and visualised with electron microscopy and their stabilityin elevated concentrations of salt and on dilution and storage forextended periods (Weissig et al. (2001) S. T. P. Pharma Sciences 11:91-96).

Examples of dequalinium and its analogues are compounds of the formula(IV):

wherein:

-   p and q are the same or different and each is an integer from 1 to    12;-   A is a bond or is selected from naphthalene, biphenyl, terphenyl,    phenanthrene, fluorene, stilbene, a group C₆H₄(CH₂)_(r)C₆H₄, a group    C₆H₄—C≡C—C₆H₄, a pyridine-2,6-diyl-bis(benzene-1,4-diyl) group, a    group CH═CH—(CH₂)_(s)—(CH═CH)_(t)—; and a group    C≡C—(CH₂)_(u)—(C≡C)_(v)—; wherein r is 0-4, s is 0 to 4, t is 0 or    1; u is 0-4 and v is 0 or 1;-   R⁸, R⁹ and R¹⁰ are the same or different and are each selected from    hydrogen; C₁₋₄ alkyl optionally substituted with one or more    fluorine atoms: C₁₋₄ alkoxy optionally substituted with one or more    fluorine atoms; nitro; amino; mono- and di-C₁₋₄ alkylamino; halogen,    phenyl-C₁₋₂ alkyl wherein the phenyl moiety is optionally    substituted with one or two methoxy, methyl or halogen substituents;-   ureido and guanidinyl; or R⁹ and R¹⁰ link together to form an    alkylene chain (CH₂)_(w) wherein w is 3 to 5; and R^(8a), R^(9a) and    R^(10a) are the same or different and are each selected from    hydrogen; C₁₋₄ alkyl optionally substituted with one or more    fluorine atoms: C₁₋₄ alkoxy optionally substituted with one or more    fluorine atoms; nitro; amino; mono- and di-C₁₋₄ alkylamino; halogen,    phenyl-C₁₋₂ alkyl wherein the phenyl moiety is optionally    substituted with one or two methoxy, methyl or halogen substituents;    ureido and guanidinyl; or R^(9a) and R^(10a) link together to form    an alkylene chain (CH₂)_(w) wherein w is 3 to 5.

Within formula (IV), one subset of compounds is the subset in which A isa bond, a group CH═CH—(CH₂)_(s)—(CH═CH)_(t)—; or a groupC≡C—(CH₂)_(u)—(C≡C)_(v)—. Within this sub-set, preferably A is a bond,i.e. there is a saturated alkylene chain extending between the nitrogenatoms of the two quinoline rings.

When A is a bond, typically the sum of p and q is in the range from 3 to22, preferably in the range from 6 to 20, and more preferably from 8 to18. Particular examples are compounds in which p+q=8, or p+q=9, orp+q=10, or p+q=11, or p+q=12, or p+q=13, or p+q=14, or p+q=15, orp+q=16, or p+q=17 or p+q=18.

In each of the foregoing embodiments and subsets of compounds, R⁸ andR^(8a) are preferably each selected from hydrogen; C₁₋₄ alkoxy; nitro;amino; mono- and di-C₁₋₄ alkylamino; and guanidinyl.

More preferably, R⁸ and R^(8a) are each selected from hydrogen; C₁₋₄alkoxy; amino and guanidinyl.

Still more preferably, R⁸ and R^(8a) are each selected from methoxy andamino.

In one embodiment, R⁸ and R^(8a) are both amino.

In another embodiment, R⁸ and R^(8a) are both methoxy.

In another embodiment, R⁸ and R^(8a) are both guanidinyl.

In each of the foregoing embodiments and subsets of compounds,preferably:

-   R⁹ is hydrogen;-   R^(9a) is hydrogen;-   R¹⁰ is selected from hydrogen; amino; and C₁₋₄ alkyl optionally    substituted with one or more fluorine atoms;-   R^(10a) is selected from hydrogen; amino; and C₁₋₄ alkyl optionally    substituted with one or more fluorine atoms;-   or R⁹ and R¹⁰ link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5: and/or-   R^(9a) and R^(10a) link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5.

More preferably:

-   R⁹ is hydrogen;-   R^(9a) is hydrogen;-   R¹⁰ is selected from hydrogen; amino; methyl and trifluoromethyl;-   R^(10a) is selected from hydrogen; amino; methyl and    trifluoromethyl;-   or R⁹ and R¹⁰ link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5: and/or-   R^(9a) and R^(10a) link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5.

In one particularly preferred group of compounds within formula (IV):

-   A is a bond;-   the sum of p and q is in the range from 8 to 18;-   R⁸ and R^(8a) are each selected from hydrogen; C₁₋₄ alkoxy; amino    and guanidinyl;-   R⁹ is hydrogen;-   R^(9a) is hydrogen;-   R¹⁰ is selected from hydrogen; amino; and C₁₋₄ alkyl optionally    substituted with one or more fluorine atoms;-   R^(10a) is selected from hydrogen; amino; and C₁₋₄ alkyl optionally    substituted with one or more fluorine atoms;-   or R⁹ and R¹⁰ link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5: and/or-   R^(9a) and R^(10a) link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5.

In another particularly preferred group of compounds within formula(IV):

-   A is a bond;-   the sum of p and q is in the range from 8 to 18;-   R⁸ and R^(8a) are each guanidinyl;-   R⁹ is hydrogen;-   R^(9a) is hydrogen;-   R¹⁰ is selected from hydrogen; amino; and C₁₋₄ alkyl optionally    substituted with one or more fluorine atoms;-   R^(10a) is selected from hydrogen; amino; and C₁₋₄ alkyl optionally    substituted with one or more fluorine atoms;-   or R⁹ and R¹⁰ link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5: and/or-   R^(9a) and R^(10a) link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5.

In another particularly preferred group of compounds within formula(IV):

-   A is a bond;-   the sum of p and q is in the range from 8 to 18;-   R⁸ and R^(8a) are each selected from hydrogen; C₁₋₄ alkoxy and    amino;-   R⁹ is hydrogen;-   R^(9a) is hydrogen;-   R¹⁰ is selected from hydrogen; amino; and C₁₋₄ alkyl optionally    substituted with one or more fluorine atoms;-   R^(10a) is selected from hydrogen; amino; and C₁₋₄ alkyl optionally    substituted with one or more fluorine atoms;-   or R⁹ and R¹⁰ link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5: and/or-   R^(9a) and R^(10a) link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5.

Within this group, more preferred compounds are those in which:

-   A is a bond;-   the sum of p and q is in the range from 8 to 18;-   R⁸ and R^(8a) are each selected from hydrogen; methoxy and amino;-   R⁹ is hydrogen;-   R^(9a) is hydrogen;-   R¹⁰ is selected from hydrogen; amino; methyl; and trifluoromethyl;-   R^(11a) is selected from hydrogen; amino; methyl; and    trifluoromethyl;-   or R⁹ and R¹⁰ link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5: and/or-   R^(9a) and R^(10a) link together to form an alkylene chain (CH₂)_(w)    wherein w is 3 to 5.

Within formula (IV), one preferred group of compounds may be representedby the formula (V):

wherein:

-   r is an integer from 2 to 24;-   R⁸, R⁹ and R¹⁰ are the same or different and are each selected from    hydrogen; C₁₋₄ alkyl optionally substituted with one or more    fluorine atoms: C₁₋₄ alkoxy optionally substituted with one or more    fluorine atoms; nitro; amino; mono- and di-C₁₋₄ alkylamino; halogen,    phenyl-C₁₋₂ alkyl wherein the phenyl moiety is optionally    substituted with one or two methoxy, methyl or halogen substituents;    ureido and guanidinyl; or R⁹ and R¹⁰ link together to form an    alkylene chain (CH₂)_(w) wherein w is 3 to 5; and R^(8a), R^(9a) and    R^(10a) are the same or different and are each selected from    hydrogen; C₁₋₄ alkyl optionally substituted with one or more    fluorine atoms: C₁₋₄ alkoxy optionally substituted with one or more    fluorine atoms; nitro; amino; mono- and di-C₁₋₄ alkylamino; halogen,    phenyl-C₁₋₂ alkyl wherein the phenyl moiety is optionally    substituted with one or two methoxy, methyl or halogen substituents;    ureido and guanidinyl; or R^(9a) and R^(10a) link together to form    an alkylene chain (CH₂)_(w) wherein w is 3 to 5;-   provided that:-   (i) when R¹⁰ and R^(10a) are both hydrogen or are both methyl, and    R⁹ and R^(9a) are both hydrogen, then at least one of R⁸ and R^(8a)    is other than hydrogen, amino or dimethylamino; and-   (ii) when R⁹ and R¹⁰ link together to form an alkylene chain    (CH₂)_(w) wherein w is 4 and R^(9a) and R^(10a) link together to    form an alkylene chain (CH₂)_(w) wherein w is 4, then at least one    of R⁸ and R^(8a) is other than amino.

Preferably, r is an integer from 8 to 20, more preferably 10 to 18, forexample any one of 10, 12, 13, 14, 15, 16, 17 and 18.

In one group of compounds within formula (V), R⁸ and R^(8a) are selectedfrom methoxy and guanidinyl. Within this group of compounds, R⁹ andR^(9a) typically are both hydrogen and R¹⁰ and R^(10a) typically areselected from hydrogen, methyl, trifluoromethyl and amino.

In another group of compounds within formula (V), R⁸ and R^(8a) areselected from hydrogen, amino, mono- and di-C₁₋₄ alkylamino; methoxy andguanidinyl; R⁹ and R^(9a) are both hydrogen and R¹⁰ and R^(10a) are bothtrifluoromethyl.

In another embodiment, the analogue may be 10,10′-(decane-1,10-diyl) bis(9-amino-1,2,3,4-tetrahydroacridinium) dichloride (FIG. 2). Thiscompound is referred to in this specification as Compound 7 because ofits original notation in Galanakis et al. (J. Med. Chem. (1995) 35:3536-3546) where it was being investigated as a potential inhibitor ofcalcium channels. The compound has subsequently been tested as an agentfor delivery of DNA therapeutics to mitochondria (Weissig et al. (2001)S. T. P. Pharma Sci. 11: 91-96) in which publication the advantageousstability of the complexes formed by the Compound 7 analogue werereported. A further paper (Weissig et al. (2006) J. Liposome Res. 16:249-264) reported that the Compound 7 analogue has lower toxicity invitro compared to dequalinium.

As analogues of dequalinium with longer alkyl chains showed even lowertoxicity, analogues with different chain lengths were considered(Weissig et al. (2006) J. Liposome Res. 16: 249-264). Thus, preferably,the alkyl chain of the dequalinium analogue has between 8 and 14 methylgroups, but with examples of chains containing as few as 3 methyl groups(Galankis et al (1996) J. Med. Chem. 39: 3592-3595) and other lipophiliccations containing as many as 36 methyl groups in the alkyl chain (Eatonet al. (2000) Angew. Chem. Int. Ed. 39: 4063-4067). More preferably, thealkyl chain has 10 or 12 methyl groups. As a result, an analogue ofCompound 7 was designed with a 12 methyl groups in the alkyl chain,referred to herein after as Compound 7_12.

Another suitable analogue is 10,10′-(dodecane-1,12-diyl) bis(9-amino-1,2,3,4-tetrahydroacridinium) dichloride (FIG. 3) denotedhereinafter as Compound 7_12. Compound 7_12 is derived from Compound 7and has a C12 alkyl chain, rather than the C10 alkyl chain of Compound7.

Other suitable analogues of dequalinium include:

-   1-decanyl-2-methyl-4-aminoquinolinium iodide-   1-butyl-2-methyl-4-aminoquinolinium iodide-   1,1,1-triethyl-1-(10-iododecan-1-yl)ammonium iodide-   1-[1-(N,N,N-triethylammonium-1-yl)-2-methyl-4-aminoquinolinium    diiodide-   1,1′-(decane-1,10-diyl)bis(4-aminopyridinium) diiodide-   1-(4-pentyn-1-yl)-4-aminopyridinium chloride-   1,1′-(deca-4,6-diyne-1,10-diyl)bis(4-aminopyridinium) dichloride    dehydrate-   2,2′-N,N′-(decane-1,10-diyl)bis(2,4-diaminopyridine) [compound 8]-   2,2′-N,N′-(decane-1,10-diyl)bis(2-aminopyridine)-   2,2′-N,N′-(decane-1,10-diyl)bis(1-methyl-2-aminopyridinium) diiodide-   1-(4-pentyn-1-yl)-2-methyl-4-aminoquinolinium iodide-   1,1′-(deca-4,6-diyne-1,10-diyl)bis(4-amino-2-methylquinolinium)    diiodide hydrate-   1,1′-(decane-1,10-diyl)bis(quinolinium) diiodide-   1,1′-(decane-1,10-diyl)bis(9-amino-1,2,3,4-tetra-hydroacridinium)    dibromide hydrate-   2,2′-(decane-1,10-diyl)bis(quinoline)-   2,2′-(decane-1,10-diyl)bis(1-methylquinolinium) diiodide hydrate-   2,2′-(decane-1,10-diyl)bis(4-methoxyquinoline)-   2,2′-(decane-1,10-diyl)bis(1-methyl-4-methoxyquinolinium) diiodide-   2,2′-(dodecane-1,12-diyl)bis(1-methylquinolinium) diiodide-   2,2′-(decane-1,10-diyl)bis(isoquinolinium) diiodide-   1,1′-(decane-1,10-diyl)bis(4-bromoisoquinolinium) diiodide-   1,1′-(decane-1,10-diyl)bis(1H-benzimidazole)-   1,1′-(decane-1,10-diyl)bis(3-methylbenzimidazolium) diiodide    hemihydrate-   1,1′-(decane-1,10-diyl)bis(2-methylbenzimidazole)-   1,1′-(decane-1,10-diyl)bis(2,3-dimethylbenzimidazolium) diiodide-   1,10-bis[N-(acridin-9-yl)amino]decane dihydrochloride dihydrate-   1,1′-(1,10-Decanediyl)bis[4-amino-2-methyl quinolinium] diiodide-   1,1′-(1,10-Decanediyl)bis[4-aminoquinolinium] diiodide-   1,1′-(1,10-Decanediyl)bis[4-N,N,dimethylaminoquinolinium] diiodide-   1,1′-(1,10-Decanediyl)bis[2-methylquinolinium] diiodide-   1,1′-(1,10-Decanediyl)bis[quinolinium] diiodide-   1,6-Bis[N-(1-methylquinolinium-2-methyl)amino] hexane diiodide-   1,1′-(1,10-Decanediyl)bis[1-amino isoquinolinium] diiodide-   1,1′-(1,10-Decanediyl)bis[2-methylbenzoxazolium] diiodide-   1,1′-(1,10-Decanediyl)bis[2-methylbenzothiazolium] diiodide-   1,1′-(1,10-Decanediyl)bis[2-amino-1-methylbenzimidazolium] diiodide-   1,1′-[(E)-5-Decene-1,10-diyl]bis[4-amino-2-methylquinolinium],    diiodide-   1,1′-[(Z)-5-Decene-1,10-diyl]bis[4-amino-2-methylquinolinium],    diiodide-   1,1′-(1,12-Dodecanediyl)bis[4-amino-2-methylquinolinium], diiodide-   1,1′-(1,14-Tetradecanediyl)bis[4-amino-2-methylquinolinium],    diiodide-   1,1′-(1,16-Hexadecanediyl)bis[4-amino-2-methylquinolinium], diiodide-   N-Decyl-4-aminoquinaldinium Iodide-   1,1′-[Biphenyl-3,3′-diylbis(methylene)]-bis(4-aminoquinolinium)-   Dibromide Hydrate (4),    1,1′-[Biphenyl-4,4′-diylbis(methylene)]bis(4-aminoquinolinium)    Ditrifluoroacetate-   1,1′-(Phenanthrene-3,6-diylbis(methylene)]bis(4-aminoquinolinium)    Dibromide Hydrate Ethanoate-   1,1′-[Fluorene-2,7-diylbis(methylene)]-bis(4-aminoquinolinium)    Ditrifluoroacetate-   1,1′-[Methylenebis(benzene-1,4-diylmethylene)]bis(4-aminoquinolinium)    Dibromide Hydrate-   1,1′-[Ethylenebis-(benzene-1,4-diylmethylene)]bis(4-aminoquinolinium)    Dibromide Hydrate-   (Z)-1,1′-[Stilbene-4,4′-diylbis(methylene)]-bis(4-aminoquinolinium)    Dibromide-   Sesquihydrate-   (E)-1,1′-[Stilbene-4,4′-diylbis(methylene)]bis-(4-aminoquinolinium)    Dibromide Dihydrate-   1,1′-[Ethyne-1,2-diylbis(benzene-1,4-diylmethylene)]bis(4-aminoquinolinium)    Dibromide Sesquihydrate-   1,1′-[Propane-1,3-diylbis(benzene-1,4-diylmethylene)]bis(4-aminoquinolinium)    Dibromide Hemihydrate Ethanoate-   1,1′-[Pyridine-2,6-diylbis(benzene-1,4-diylmethylene)]-bis(4-aminoquinolinium)    Dibromide Hydrate-   1,1′-[Butane-1,4-diylbis(benzene-1,4-diylmethylene)]bis-(4-aminoquinolinium)    Dibromide Hydrate-   1,1′-[1,1:4′,1″-Terphenyl-4,4″-diylbis(methylene)]bis(4-aminoquinolinium)    Dibromide Trihydrate-   1,1′-[Naphthalene-2,6-diyl(bis(methylene)]bis(4-aminoquinolinium)    Dibromide Hydrate-   1,1′-[Benzene-1,4-diylbis(methylene)]-bis(4-aminoquinolinium)    Dibromide Dihydrate-   1,1′-[Benzene-1,3-diylbis(methylene)]bis(4-aminoquinolinium)    Dibromide Hemihydrate-   1,1′-(Propane-1,3-diyl)bis(4-aminoquinolinium) diiodide-   1,1′-(Butane-1,4-diyl)bis(4-aminoquinolinium) diiodide-   1,1′-(Pentane-1,5-diyl)bis(4-aminoquinolinium) diiodide-   1,1′-(Hexane-1,6-diyl)bis(4-aminoquinolinium) diiodide-   1,1′-(Octane-1,8-diyl)bis(4-aminoquinolinium) diiodide-   1,1′-(Dodecane-1,12-diyl)bis(4-aminoquinolinium) dibromide    hemihydrate-   1,10-Bis[N-(2-methylquinolin-4-yl)amino]decane-   1,12-Bis[N-(2-methylquinolin-4-yl)amino]dodecane-   1,10-Bis[(2-methylquinolin-4-yl)amino]decane-   1,12-Bis[(2-methylquinolin-4-yl)amino]dodecane-   1,10-Bis(N-quinolin-4-ylamino)decane-   4,4′-[Decane-1,10-diylbis(oxy)]bis[quinoline]-   4,4′-[Decane-1,10-diylbis(thio)]bis[quinoline]-   4,4′-Dodecane-1,12-diylbis[quinoline]-   1,8-Bis(N-quinolin-4-yldiamino)octane-   1,8-Bis[N-(1-methylquinolinium-4-yl)amino]octane Diiodide Hydrate-   1,10-Bis[N-(1-methylquinolinium-4-yl)amino]decane Diiodide-   4,4′-[Decane-1,10-diylbis(oxy)]bis[1-methylquinolinium] Diiodide-   4,4′-[Decane-1,10-diylbis(thio)]bis[1-methylquinolinium] Diiodide    Hydrate (10).-   1,1′-Dimethyl-4,4′-dodecane-1,12-diylbis[quinolinium] Diiodide-   4,4′-Decane-1,10-diylbis[quinoline]-   1,1′-Dimethyl-4,4′-decane-1,10-diylbis[quinolinium] Diiodide-   1,10-Bis[N-(1-benzylquinolinium-4-yl)amino]decane Dibromide-   1,10-Bis[N-(1-benzyl-2-methylquinolinium-4-yl)amino]-decane    Bis(trifluoroacetate)-   1,12-Bis[N-(1-benzyl-2-methylquinolinium-4-yl)amino]-dodecane    Bis(trifluoroacetate)-   1-[N-(1-Benzyl-2-methylquinolinium-4-yl)amino]-10-[N-(2-methylquinolinium-4-yl)amino]decane    Bis(trifluoroacetate)-   1-[N-(1-Benzyl-2-methylquinolinium-4-yl)amino]-12-(N′-(2-methylquinolinium-4-yl)amino]dodecane    Bis(trifluoroacetate)-3,5-Dimethoxybenzyl iodide-   1,10-Bis[N-[1-(3,5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]decane    Bis(trifluoroacetate)-   1-[N-[1-(3,5-Dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N-(2-methylquinolinium-4-yl)amino]decane    Bis(trifluoroacetate)-   1,1′-(3-Iodopropylidene)bis[benzene]-   1,10-Bis[N-[1-(3,3-diphenylprop-1-yl)-2-methylquinolinium-4-yl]amino]decane    Bis(trifluoroacetate)-   4,7-Dichloro-1-methylquinolinium Iodide-   1,10-Bis[N-(7-chloro-1-methylquinolinium-4-yl)amino]-decane Diiodide    Dihydrate.

Dequalinium and its salts are commercially available, for example from(Sigma Aldrich). Methods of making suitable analogues are described inWO 97/48705, Galanakis et al. (1995) J. Med. Chem. 38: 595-606,Galanakis et al (1995) J. Med. Chem. 38: 3536-3546 and Galanakis et al(1996) J. Med. Chem. 39: 3592-3595, Abeywickrama et al. (2006)Bioorganic Medicinal Chem. 14: 7796-7803, Qin et al. (2000) J. Med.Chem. 43: 1413-1417, Campos Rosa et al (1996) J. Med. Chem. 39:4247-4254, the contents of which are incorporated herein by reference.In particular, the synthesis of Compound 7 is described in Galanakis etal. (1995) supra and the synthesis of Compound 7_12 may be derivedtherefrom. The compounds of formula (IV) and (V) may be prepared bymethods analogous to the known methods for preparing dequalinium, asdescribed and referenced above.

For example, compounds of the formulae (IV) and (V) can be prepared bythe reaction of a quinoline compound of the formula (VI):

with a compound of the formula I—(CH₂)_(r)—I. The reaction is typicallycarried out at an elevated temperature, for example in the range 120° C.to 160° C., e.g. at around 150° C.

Quinoline compounds of the formula (VI) are commercially available orcan be made by standard methods well known to the skilled person ormethods analogous thereto, see for example Advanced Organic Chemistry byJerry March, 4^(th) Edition, John Wiley & Sons, 1992, Organic Syntheses,Volumes 1-8, John Wiley, edited by Jeremiah P. Freeman (ISBN:0-471-31192-8), 1995, Fiesers' Reagents for Organic Synthesis, Volumes1-17, John Wiley, edited by Mary Fieser (ISBN: 0-471-58283-2), andHandbook of Heterocyclic Chemistry, A. R. Katritzky et al, 3^(rd)Edition, Elsevier, 2010.

Compounds of the formula (VI) wherein R⁸ is amino and R⁹ and R¹⁰ linktogether to form an alkylene chain (CH₂)_(w) can be prepared by means ofthe following reaction sequence:

The amino group may then be converted into other functional groups bystandard methods, for example by Diazotisation followed by a Sandmeyerreaction.

The nucleic acid sequence may be an oligonucleotide sequence or apolynucleotide sequence. An oligonucleotide sequence is generallyrecognised as a linear sequence of up to 20 nucleotides joined byphosphodiester bonds, while a polynucleotide sequence typically has morethan 20 nucleotides and maybe single or double stranded with varyingamounts of internal folding. The backbone may also be modified toincorporate synthetic chemistries known either to reduce the charge ofthe molecule or increase its stability in biological fluids. Examples ofthese include peptide nucleic acids (PNA), linked nucleic acids (LNA),morpholino oligonucleotides and phosphorothioate nucleotides andcombinations of these.

In one embodiment, the nucleic acid sequence comprises the sequence of anative cellular binding site for a transcription factor. Such a sequenceis referred to as a transcription factor decoy (TFD). Preferably thedecoy comprises the sequence of a bacterial Sig binding site.Alternatively, the decoy comprises the sequence of a bacterial Furbinding site.

Examples of suitable TFD sequences are provided in SEQ ID NOs: 11, 12,13, 32, 33, 39, 40, 41, 42, 43 and 44.

TFDs are effective at nanomolar concentrations and have been effectiveat preventing growth of bacteria in vitro and in vivo at concentrationsas low as 1 nM, although it is anticipated that against certain bacteriaand in more complex settings, such as in a patient higher concentrationsmay be needed. Hence, a preferred range would therefore be between about10 to 100 nM, and up to around 1 μM. It will be appreciated that therange encompasses concentrations in between about 10 nM and 1 μM, suchas 20 nM, 20 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 150 nM, 200nM, 500 nM, 750 nM, and intermediates thereof, for example 27.2 nM.

Where the delivery moiety is a compound of any of formulae (I) to (V)such as dequalinium or an analogue thereof, complexes are formed betweenthe nucleic acid and the compound (e.g. dequalinium or an analoguethereof) using different ratios of both. The ratio is commonly referredto as the N/P ratio (for example see Zhao et al. (2007)Biomacromolecules 8: 3493-3502), which defines the number of positivelyNitrogen atoms in the delivery molecule per negatively charged Phosphateatom in the nucleic acid, or per nucleotide when no phosphate atoms arepresent. Typically complexes are formed between dequalinium (or itsanalogues) and TFDs at N/P ratios between 0.1 and 1 (which is sufficientto achieve charge neutralisation). It will be appreciated that thepresent invention encompasses ratios in between 0.1 and 1, such as 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and intermediates thereof, e.g. 0.23.

Complexes capable of transfecting bacteria in vitro are well toleratedin animal studies. Furthermore, the components of such complexes may beused at concentrations below their known Maximum Tolerated Dose (MTD).Maximum Tolerated Dose (MTD) is the highest daily dose that does notcause overt toxicity. MIC can be estimated by animal studies as theamount of compound that, when administered to a group of test animals,has no measurable affect on long term survivability. For example,administering a complex containing 1 μM of a 100 nucleotide TFD with anN/P ratio of 1 would give a dose of approximately 3 mg/kg dequaliniumanalogue. It will be appreciated that this dose of dequalinium analogueis substantially below the MTD of dequalinium. The analogues ofdequlanium, such as Compound 7, show less in vitro toxicity thandequalinium (Weissig et al. (2006) J. Liposome Res. 16: 249-264).Because dequalinium has an MTD of 15 mg/kg in mice (Gamboa-Vujicic etal. (2006) J. Pharm. Sci. 82: 231-235) it is predicted that thecomplexes should be well tolerated. It is within the reasonable skilland knowledge of the skilled person to calculate and prepare suitableconcentrations.

In an alternative embodiment, the antibacterial complex comprises anucleic acid sequence and an antibacterial peptide. The termantibacterial peptide includes and encompasses antimicrobial peptides,cell penetrating peptides, non-ribosomally synthesised peptides andglycopeptides.

Antimicrobial peptides (AMPs; also called host defense peptides) areancient and natural antimicrobials that are diverse and widespread. Theyare an evolutionarily conserved component of the innate immune responseand are found among all classes of life. Fundamental differences existbetween prokaryotic and eukaryotic cells that may represent targets forantimicrobial peptides. These peptides are potent, broad spectrumantibiotics which demonstrate potential as novel therapeutic agents.Antimicrobial peptides have been demonstrated to kill Gram-negative andGram-positive bacteria (including strains that are resistant toconventional antibiotics), mycobacteria (including Mycobacteriumtuberculosis), enveloped viruses and fungi. Unlike the majority ofconventional antibiotics, it appears that antimicrobial peptides mayalso have the ability to enhance immunity by functioning asimmunomodulators.

Antimicrobial peptides are generally between 12 and 50 amino acids.These peptides include two or more positively charged residues providedby arginine, lysine or, in acidic environments, histidine, and a largeproportion (generally >50%) of hydrophobic residues. The secondarystructures of these molecules follow four themes, including i)α-helical, ii) β-stranded due to the presence of two or more disulfidebonds, iii) β-hairpin or loop due to the presence of a single disulfidebond and/or cyclisation of the peptide chain, and iv) extended. Many ofthese peptides are unstructured in free solution and fold into theirfinal configuration upon partitioning into biological membranes. Thepeptides contain hydrophilic amino acid residues aligned along one sideand hydrophobic amino acid residues aligned along the opposite side of ahelical molecule. This amphipathicity of the antimicrobial peptidesallows the peptides to partition into the membrane lipid bilayer. Thesepeptides have a variety of antimicrobial activities ranging frommembrane permeabilisation to action on a range of cytoplasmic targets.

The modes of action by which antimicrobial peptides kill bacteria isvaried and includes disrupting membranes, interfering with metabolism,and targeting cytoplasmic components. The initial contact between thepeptide and the target organism would be electrostatic, as mostbacterial surfaces are anionic. Their amino acid composition,amphipathicity, cationic charge and size allow them to attach to andinsert into membrane bilayers to form pores by ‘barrel-stave’, ‘carpet’or ‘toroidal-pore’ mechanisms. Once the cell has been penetrated, thepeptides bind to intracellular molecules which are crucial to cellliving, thereby inhibiting cell wall synthesis, altering the cytoplasmicmembrane, activating autolysin, inhibiting DNA, RNA, and proteinsynthesis, and inhibiting certain enzymes. However, in many cases, theexact mechanism of killing is not known. In contrast to manyconventional antibiotics, these peptides appear to be bacteriocidal(bacteria killer) instead of bacteriostatic (bacteria growth inhibitor).

In the competition of bacterial cells and host cells with theantimicrobial peptides, antimicrobial peptides will preferentiallyinteract with the bacterial cell to the mammalian cells, which enablesthem to kill microorganisms without being significantly toxic tomammalian cells. Since the surface of the bacterial membranes is morenegatively charged than mammalian cells, antimicrobial peptides willshow different affinities towards the bacterial membranes and mammaliancell membranes.

It is well known that cholesterol is normally widely distributed in themammalian cell membranes as a membrane stabilizing agents but is absentin bacterial cell membranes. The presence of these cholesterols willalso generally reduce the activities of the antimicrobial peptides, dueeither to stabilisation of the lipid bilayer or to interactions betweencholesterol and the peptide. Thus, the cholesterol in mammalian cellswill protect the cells from attack by the antimicrobial peptides.

In addition, the transmembrane potential is well-known to affectpeptide-lipid interactions. A negative transmembrane potential existsbetween the outer leaflet to the inner leaflet of a cell membrane. Thisinside-negative transmembrane potential facilitates membranepermeabilisation probably by facilitating the insertion of positivelycharged peptides into membranes. By comparison, the transmembranepotential of bacterial cells is more negative than that of normalmammalian cells, so bacterial membrane will be prone to be attacked bythe positively charged antimicrobial peptides.

As discussed above, AMPs are a unique and diverse group of molecules,which are divided into sub-groups on the basis of their amino acidcomposition and structure. A database of many of the known AMPs can befound at http://www.bbcm.units.it/˜tossi/pag1.htm. Other groups ofpeptides with anti-infective properties include the non-ribosomalpeptides, examples of which include gramicidin, and as a sub-group thosewith glycopeptides antibiotics, where peptides (which are commonlycyclic) are glycosylated. Examples of these include Polymyxin.

The present inventors have made a functional classification of all ofthese peptides, which are termed Anti-Bacterial Peptides (ABPs) herein,to distinguish them from AMPs, based on the mechanism of bacterialkilling, and include peptides derived from other classes ofantibacterials such as cell-penetrating peptides, non-ribosomallysynthesised peptides and glycopeptides. It will be appreciated that theinvention encompasses naturally occurring and non-naturally occurring,synthetic peptides.

Class I. ABPs that are membrane active (Polymyxin, gramicidin) andaffect entry by causing sufficient damage to punch holes in the outermembrane and allow extrusions (or ‘blebs’) of the bacterial innermembrane to form, through which large molecules can pass. Several ofthese peptides are used in the clinic but there are concerns abouttoxicity as they damage eukaryotic membranes as well.

The rate of resistance against antibacterial peptides is remarkably lowand these peptides are widespread in nature. As this class ispredominantly cationic, resistance mechanisms would take the form ofchanges to charge density on the outer membrane of bacteria, and suchchanges have been seen in the lab. Hence, the relative low incidence ofresistance may reflect the fact that the ABPs are not that effective,rather than being difficult to resist.

Class II. This is a much smaller class. These ABPs do not damage themembrane but instead have intracellular targets. Thus, the peptides musttranslocate through the bacterial membranes to reach their targets. As aresult, they are markedly less toxic than Class I ABPs as they do notdamage eukaryotic membranes causing haemolysis etc. Although they arecationic, their ability to cross membranes is not expected to be solelypredicated on their charge but other broader structural properties,which largely remain undefined. As consequence, resistance mechanismsare thought less likely to occur as such mechanisms would need to alterthe hydrophobic nature of the bacterial membranes themselves.

An example of an ABP that is capable of translocation is Buforin and atruncated form Buforin II (BF2). This peptide shows (weak) broadspectrum activity against pathogenic bacteria (Park et al. (2000) Proc.Natl. Acad. Sci. USA 97: 8245-8250). It has been used to translocateeukaryotic membranes and even to deliver a 28 kDa peptide (GFP) to humancell lines (Takeshima et al. (2003) J. Biol. Chem. 278: 1310-1315),although this was via endocytosis and probably due to the cationicnature of the peptide.

The ABP may be a naturally occurring peptide, such as Gramicidin, orBuforin Alternatively, the ABP may be a peptidomimetic or a syntheticvariant of a naturally occurring peptide, such as Buforin II orPolymyxin nonapeptide.

Examples of antimicrobial peptides with the ability to permeabilisebiological membranes are provided in Papagianni et al ((2003)Biotechnol. Adv. 21: 465-499) and include defensins, pleuricidins,magainins, dermaseptins, apidaecins, cecropins, microcins and pediocins.

Examples of antibacterial peptides with the ability to permeabilisebiological membranes are provided in Varra et al ((1992) Microbiol. Rev.56: 395-411) and include the lantibiotics, glycopeptide antibiotics,cationic polypeptides such as polylysine and polyarginine.

Types and characteristics of ABPs are summarised in Table 1:

Type Characteristic ABPs Anionic rich in Maximin H5 from amphibians,peptides glutamic and Dermcidin from humans aspartic acids Linearcationic lack in Cecropins, andropin, moricin, α-helical cysteineceratotoxin and melittin from insects, peptides Magainin, dermaseptin,bombinin, brevinin-1, esculentins and buforin II from amphibians, CAP18from rabbits, LL37 from humans Catioinic rich in proline, abaecin,apidaecins from honeybees, peptide arginine, prophenin from pigs,indolicidin from enriched for phenylalanine, cattle. specific aminoglycine, acid tryptophan Anionic and contain 1~3 1 bond: brevinins, 2bonds: cationic peptides disulphide protegrin from pig, tachyplesinsfrom that contain bond horseshoe crabs, 3 bonds: defensins cysteine andform from humans, more than 3: disulfide bonds drosomycin in fruit fliesAnionic and Generated by Cascocidin I from human casein, cationicpeptide peptidic lactoferricin from lactoferrin, fragments of cleavageantimicrobial domains from human larger proteins haemoglobin or lysozyme

The ABP may be linked to the nucleic acid by electrostatic or covalentlinkages. In particular, where the mechanism of killing of the ABP isvia intracellular targeting, rather than membrane permeabilisation, thecomplex ideally includes covalent linkage, such as a suitable linker orcross-linker between the nucleic acid sequence and the ABP. An exampleof a suitable linker is one that couples a carboxyl group to a primaryamine. For example, a suitable linker may be EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride).

EDC is known for its use as a carboxyl activating agent for the couplingof primary amines to yield amide bonds and a common use for thiscarbodiimide is protein cross-linking to nucleic acids.

Although ABPs have antimicrobial activity on their own, it has beenfound that such peptides act synergistically when in combination withTFDs to prevent bacterial growth. Some ABPs have a bacteriostatic effectand others rapidly kill on contact. It has been found that sub-lethalconcentrations of the peptides allow entry of TFDs. The peptides stressthe bacteria making them more vulnerable to TFDs, which then blockstress causing growth stasis or cell death.

The complexes are prepared so that the ABPs are at a concentration thatis typically 5-10 fold less than their Minimum Inhibitory Concentration(MIC). The MIC of a compound is the minimum concentration of thatcompound to prevent visible growth of the bacteria. Typically the MIC isdetermined by a dilution method where inoculated cultures of bacteriaare incubated overnight with a series of concentrations of the compoundand the one that prevents growth is taken as the MIC. It is within thereasonable skill and knowledge of the skilled person to calculate andprepare suitable concentrations.

In a yet further embodiment, the antibacterial complex of the presentinvention comprises a) a nucleic acid sequence, b) a quaternary aminecompound or bis-aminoalkane, or unsaturated derivative thereof, whereinthe amino component of the aminoalkane is an amino group forming part ofa heterocyclic ring, and c) an antibacterial peptide.

Expressed in another way, the antibacterial complex of the presentinvention comprises a) a nucleic acid sequence, b) a quarternaryderivative of quinoline or acridine, and c) an antibacterial peptide.

In a particularly preferred embodiment, the antibacterial complexcomprises a) a transcription factor decoy, b) a dequalinium analogue andoptionally, c) an antimicrobial peptide.

In a yet further aspect, the present invention residues in use of thecomplexes of the invention in a suitable formulation for the treatmentof one or more bacterial infections.

In particular, the invention provides a method for treating bacterialinfection in a subject comprising administering a nucleic acid sequenceformulated as described herein. The subject may be a human or animal.The invention also provides a nucleic acid sequence formulated asdescribed herein for use in medicine, e.g. for use in treating orpreventing bacterial infection in a subject, and the use of the nucleicacid sequence formulated as described herein for the manufacture of amedicament for treating bacterial infection.

The invention further relates to a pharmaceutical composition ormedicament comprising a nucleic acid sequence, a nucleic acid sequence,at least one delivery moiety, wherein the delivery moiety is selectedfrom quaternary amine compounds; bis-aminoalkanes and unsaturatedderivatives thereof, wherein the amino component of the aminoalkane isan amino group forming part of a heterocyclic ring; and an antibacterialpeptide and a physiologically acceptable carrier or excipient. Thecomposition may additionally comprise one or more antibiotic or otherantibacterial compound or composition.

The number of nucleic acid sequences needed to show a predictable effecton expression of a targeted gene and have a bacteriostatic orbacteriocidal effect can be as little as circa 5000 molecules per cell.It has been found that as many as 1,000,000 bacterial cells areefficiently killed with as little as 1 nM of TFD (WO/2010/038083),suggesting that it is sufficient to have a transfection efficiency ofless than 0.001% to achieve killing. In comparison with other nucleicacid-based strategies to tackle bacterial infections, such as antisense,this number of molecules needed to kill the cell is 100 to 1000-foldless. This partly reflects that although both antisense approaches andTFDs act to inhibit genes, TFDs act at an early step to preventtranscription whilst antisense, in the most common iteration, stericallyblocks the products of transcription: many thousands of mRNAs molecules.Secondly, the TFDs have been designed to target essential genes that arepositively induced, so need to be switched on for survival, andpositively regulated (the transcription factor drives its ownproduction). In vitro, this latter characteristic means that relativelyfew copies of the transcription factor are likely present when the geneis uninduced and so a small number of TFDs can block induction.

It may be that, in a therapeutic situation, there are more transcriptionfactors per cell, due to natural variety amongst the bacterialpopulation or the gene being already induced. In this situation it isexpected that more TFDs will be needed to see a therapeutic effect andestimate that increasing the dose by a factor of 100 (to 100 nM) orimproving the transfection efficiency (by two orders of magnitude) willbe sufficient to see a beneficial effect. Transfection may be quantifiedusing fluorescence microscopy (Zhang et al. (1996) J. Mol. Neurosci. 7:13-28).

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise as, or inaddition to active ingredient, a pharmaceutically acceptable excipientor diluent any suitable binder, lubricant, suspending agent, coatingagent, solubilising agent or other materials well known to those skilledin the art. Such materials should be non-toxic and should not interferewith the efficacy of the active ingredient. Acceptable carriers ordiluents for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remington' Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985). The precise nature ofthe carrier or other material will depend on the route ofadministration, which may be oral, or by injection, e.g. cutaneous,subcutaneous or intravenous.

The active ingredient is defined as a nucleic acid sequence, such as aTFD, complexed (or formulated) with a delivery moiety, the deliverymoiety being the delivery moiety is selected from quaternary aminecompounds; bis-aminoalkanes and unsaturated derivatives thereof, whereinthe amino component of the aminoalkane is an amino group forming part ofa heterocyclic ring; and an antibacterial peptide. In thecomplex/formulation, the quaternary amine compound, bis-aminoalkane orunsaturated derivative thereof, such as dequalinium or its analogue, isin the form of a bolasome. The term ‘bolasome’ is used in thisspecification to describe vesicles of the derivative after the compoundhas been subjected to sonication (see Weissig and Torchilin (2001) Adv.Drug Delivery Rev. 49: 127-149).

A variety of methods may be used to deliver the antibacterial complex ofthe present invention to the site of bacterial infection. Methods for invivo and/or in vitro delivery include, but are not limited to, bucchalor oral delivery, intravenous delivery, direct injection into theinfection or indirect injection (e.g. subcutaneous, intraperitoneal,intramuscular, or other injection methods), topical application, directexposure in aqueous or media solution, transfection (e.g. calciumphosphate, electroporation, DEAE-dextran based, and lipid mediated),transgenic expression (e.g. a decoy expression system delivered bymicroinjection, embryonic stem cell generation, or retroviral transfer),or any of the other commonly used nucleic acid delivery systems known inthe art. Administration may be in combination with a suitable dose ofantibiotic, with the antibiotic(s) being administered at the same timeas the nucleic acid sequence, or separately.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatine or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded. For intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, tonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as sodium chloride, Ringer'sinjection, lactated Ringer's injection, preservatives, stabilisers,buffers, antioxidants and/or other additives may be included, asrequired.

For some applications, pharmaceutical formulation may not be required.For example, the antibacterial complex of the invention may be toleratedas a pharmaceutical in its own right, without the need for excipientsand/or carriers.

Alternatively, the antibacterial complex may be suitable for use as anantibacterial disinfectant and so may be required in a suitable aqueousformat. In which instance, the complex may further comprise aqueous andorganic solvents and their combinations.

The antibacterial complex of the invention may be used to treat avariety of bacterial infections wherever they occur within the humanbody. Five general areas of bacterial infection can be described.

Respiratory tract infections are amongst the commonest, the upperrespiratory tract infections including the ears, throat, and nasalsinuses that can be treated with tropical applications or aerosolpreparations. Lower tract infections include pneumonia (which is causedby a range of bacterial pathogens, bronchitis and infectivecomplications of cystic fibrosis.

A common problem in both community and hospital practice is urinarytract infections, where the urine becomes infected and antibacterialsneed to enter the bladder, prostrate, ureter and kidneys.

The gut is vulnerable to infections, where bacteria cause disease byeither by mucosal invasion or toxin production, an example of whichincludes cholera epidemics, and when used antibiotics are eitheringested or administered intravenously.

Skin and soft tissue infections, which can be treated by topicalapplications, are common following traumatic injury or burns, whichallow colonisation and ingression of micro-organisms resulting ininfections that are both localised or have spread rapidly throughtissues. Microbes responsible for skin infections often arise fromnormal skin flora, such as Streptococcus pyogenes causing superficialskin infections (impetigo), cellulitis (more deep-seated infection thatcan spread to the blood) and necrotising fasciitis, a rapidlyprogressive infection that is often life-threatening.

Finally infections of the central nervous system, such as bacterialmeningitis, are perhaps the most challenging to treat as therapies mustpenetrate the blood-brain barrier, as too have the pathogenic bacteria.

The antibacterial complex of the present invention may be used incombination with one or more antibiotics to which the nucleic acidsequence makes the bacterial cell more sensitive, and/or with anotherantibacterial agent. Suitable antibiotics are described in co-pendingapplications WO 2009/044154 and WO 2010/038083. It will be appreciatedthat the lists provided therein may not be exhaustive. The presentinvention encompasses any suitable antibiotic or antibacterial compoundsor compositions.

The antibiotic or antibacterial compound may be administeredsimultaneously with, or before or after the antibacterial complex of theinvention. The antibiotic/antibacterial compound and antibacterialcomplex may be administered in the same or in separate compositions.Thus the invention includes combination therapies in which anantibacterial complex as identified, and/or as described, herein isadministered to a subject in combination with one or more antibiotics orother antibacterial therapies. The composition may additionally compriseone or more antibiotic or other antibacterial compounds or compositions.

Suitable TFDs encompassed by the invention and/or their targets arelisted below. The sequences provided herein illustrate single strands ofthe binding sites. However, it will be appreciated that in nature and inthe TFDs of the present invention, the sequences will be doublestranded. The complementary strands to the sequences listed herein areclearly and easily derivable, for example from Molecular Cloning: ALaboratory Manual (3^(rd) Edition), 2001 by Joseph Sambrook and DavidRussell.

WhiB7

The native WhiB7 binding site in M. smegmatis str MC2 155 comprises:

(SEQ ID NO: 1) 5′-CACCAGCCGA AAAGGCCACG GACCCGCAGT CACCCGGATC CGTGGCCATTTTTGTCGGAC CCCCCGAGAA ATCTGGTCGC AGGATCCATC AGCTCAGACA GATCAC-3′WhiB7 TFD:

(SEQ ID NO: 2) 5′ TGG CCA CGG ATC CGG GTG ACT GCG GGT CCG TGG CCT 3′FadR

The native FadR binding site in E. coli K12 comprises the sequence:

(SEQ ID NO: 3) 5′ AGTAAGTTTC GAATGCACAA TAGCGTACAC TTGTACGCCG AACAAGTCCGATCAGCCATT TAA-3′

One example of a FadR TFD sequence comprises

(SEQ ID NO: 4) 5′ TTT ATT CCG AAC TGA TCG GAC TTG TTC AGC GTA CAC GTGTTA GCT ATC CTG CGT GCT TCA 3′YycG/YycF

The native binding sites for YycF and YycG in S. aureus (in the LytM andSsa promoters) comprise:

YycF_LytM (SEQ ID NO: 5) 5′-GCTATTTTGTAATGACAATGTAATGAGTTTAGTAAAAA-3′YycF_SsaA (SEQ ID NO: 6) 5′-ATTACAAATTTGTAACAGACTTATTTTA-3′

Examples of a YycG/YycF TFD sequence include:

LytM TFD (SEQ ID NO: 7) 5′ GCT ATT TTG TAA TGA CAA TGT AAT GAG TTT AGTAAA AA 3′ SsaA TFD (SEQ ID NO: 8) 5′ ATT ACA AAT TTG TAA CAG ACT TAT TTTA 3′Sigma 54 or Sig^(B)

The native binding sites in S. aureus and K. pneumoniae comprise:

SA_sig: (SEQ ID NO: 9) 5′-TTATTATATA CCCATCGAAA TAATTTCTAA TCTTC-3′KP_sig: (SEQ ID NO: 10) 5′-CCGATAAGGG CGCACGGTTT GCATGGTTAT-3′Fur

Consensus sequences for Fur binding in S. aureus and E. coli comprise:

SA_fur: (SEQ ID NO: 11) 5′-ACT ACA AGT ACT ATT AGT AAT AGT TAA CCCTT-3′.Consensus sequence (‘Fur BOX’) as described in Horsburgh, J.Bacteriology (2001) 183:468.

EC_fur: (SEQ ID NO: 12) 5′-GATAATGATAATCATTATC-3′.Consensus sequence as described in de Lorenzo, J. Mol. Biol. (1998)283:537.

A native binding sequence in H. pylori comprises:

HP_fur: (SEQ ID NO: 13) 5′-GTT GTC CCA TAA TTA TAG CAT AAA TGA TAA TGAAAA AGT AAA-3′TcdR

A consensus binding site in C. difficile comprises:

(SEQ ID NO: 14) 5′-AAG TTT ACA AAA TTA TAT TAG AAT AAC TTT TTT A TT-3′.Consensus sequence (TcdR, where −35 and −10 boxes are underlined) asdescribed in Dupuy, Mol. Micro. (2006) 55:1196.Vfr

A consensus and two native binding sites in P. aeruginosa are:

PA_Vfr: (SEQ ID NO: 15) 5′-AAA TGT GAT CTA GAT CAC ATT T-3′.

Consensus sequence as described in Kanack, Microbiol. (2006) 55:1196.

PA_ToxA: (SEQ ID NO: 16) 5′-CACTCTGCAA TCCAGTTCAT AAATCC-3′ PA_RegA:(SEQ ID NO: 17) 5′-GTAACAGCGGAACCACTGCACAG-3′NtrC

A native binding site in K. pneumoniae comprises:

(SEQ ID NO: 18) 5′-GCTTTGCACTACCGCGGCCCATCCCTGCCCCAAAACGATCGCT-3′ArsR

Examples of a native binding sequence comprise:

HP_AmiE: (SEQ ID NO: 19) 5′-ATAATCATAA TGATTAAAGT TTTCATATTC ATTATAAATCCGTTTACACA ATTATT-3′ HP_RocF: (SEQ ID NO: 20) 5′-GAAATTGTTC TATTTATTATCCATTTGCTT ATTAATAATT GGTTGTTAAT TTTGGTTTAG A-3′Glycopeptide-resistant Consensus Sequence (GISA)

An example of the consensus sequence, found in the promoter of tcaA, aknown positive regulator of virulence (Maki, Antimicrobial AgentsChemother. (2004) 48:1953) may be used in an antibacterial complex ofthe invention, for example:

SA_TcaA: (SEQ ID NO: 21) 5′-TGAACACCTTCTTTTTA-3′AgrA

Examples of a sequences for motifs found in genes to be positivelyregulated by Agr, a regulator associated with virulence (Dunman, J.Bacteriol. (2001) 183:7341) are:

SA_Agr_2093: (SEQ ID NO: 22) 5′-AGA AAG ACA AAC AGG AGT AA-3′SA_Agr_1269: (SEQ ID NO: 23) 5′-GAA GAA ACA AAA AGC AGC AT-3′

Suitable primer sequences for a S. aureus Sig TFD are:

SAsigB FOR: (SEQ ID NO: 24) GAA GAT TAG AAA TTA TTT CGA T GGG TAT ATAATA A; and SASigB REV: (SEQ ID NO: 25 TAT TAT ATA CCC ATC GAA ATA ATTTCT AAT CTT C A.

Suitable primer sequences for a S. aureus Fhu TFD are:

SAfhu FOR: (SEQ ID NO: 26) ACT ACA AGT ACT ATT AGT AAT AGT TAA CCC TA;and SAfhu REV: (SEQ ID NO: 27) AGG GTT AAC TAT TAC TAA TAG TAC TTG TAGTA

Suitable primer sequences for an S. aureus SsaA TFD are:

SsaA FOR: SEQ ID NO: 28) ATT ACA AAT TTG TAA CAG ACT TAT TTT A; and SsaAREV: SEQ ID NO: 29 AAA ATA AGT CTG TTA CAA ATT TGT AAT A

To form a Sig dumbbell TFD (referred to as SA3 TFD), the followingphosphorylated oligonucleotides may be synthesised:

SigDB_SA3: (SEQ ID NO: 30) CTTGG TTTTT CCAAG GAA GAT TAG AAA TTA TTT CGAT GGG TAT ATA ATA; and SigDB_SA3: (SEQ ID NO: 31) P-CCG TCT TTT TGA CGGTAT TAT ATA CCC ATC GAA ATA ATT TCT AAT CTT C

The sequences of the pairs of oligonucleotides used to form a WalR_TFDare:

WalR1: (SEQ ID NO: 32) 5′-P-CTT GGT TTT TCC AAG TAA TGA ATG AGT TTA AAGCCC ATG TAA AAG GGG TAT CAG TAC-3′; and WalR2: (SEQ ID NO: 33) 5′-P-CCCTCT TTT TGA GGG GTA CTG ATA CCC CTT TTA CAT GGG CTT TAA ACT CAT TCATTA-3′.

Other suitable primers for TFDs include:

fabBf: (SEQ ID NO: 34) 5′-tct tta aat ggc tga tcg gac ttg-3′; and fabBr(SEQ ID NO: 35) 5′-agt aag ttt cga atg cac aat agc gta-3′. Ks54f: (SEQID NO: 36) P-CCG ATA AGG GCG CAC GGT TTG CAT GGT TAT A; and Ks54r: (SEQID NO: 37) P-ATA ACC ATG CAA ACC GTG CGC CCT TAT CGG A.

A consensus sequence for a WalR TFD may be TGT WAW NNN NNT GTA AW (SEQID NO: 38) where: W is A or T.

A consensus sequence for a SigB TFD may be GKT TWA NNN NNN NNN NNN NNKGGT AW (SEQ ID NO: 39) where: K is G or T; W is A or T.

An example of a KP sig TFD sequence is TGG CAC AGA TTT CGC T (SEQ ID NO:40)

A consensus sequence for a KP_Sig TFD may be TGG NNN NNN WTT TGC W (SEQID NO: 41) where W is A or T.

Such TFDs may be prepared and tested as described in co-pendingapplications WO 2009/044154 and WO 2009/044154.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described by way of non-limitingexamples and figures, in which:

FIG. 1. Chemical structure of Dequalinium.

FIG. 2. Chemical structure of 10,10′-(decane-1,10-diyl) bis(9-amino-1,2,3,4-tetrahydroacridinium) dichloride.

FIG. 3. Chemical structure of 10,10′-(dodecane-1,12-diyl) bis(9-amino-1,2,3,4-tetrahydroacridinium) dichloride.

FIG. 4. Size distribution of 100 μM sC7 bolasomes as measured by dynamiclight scattering.

FIG. 5. Size distribution of 100 μM sC7_12 bolasomes as measured bydynamic light scattering.

FIG. 6. SYBR Green-DNA binding assay to measure binding of Compound 7bolasomes to TFDs.

FIG. 7. SYBR Green-DNA binding assay to measure binding of Compound 7_12bolasomes to TFDs.

FIG. 8. SYBR Green-DNA binding assay to quantify the stability ofcomplexes formed with Compound 7 bolasomes and Sig_TFD.

FIGS. 9A & 9B. Electron micrographs of TFD complexes formed withCompound 7 bolasomes and Sig TFD.

FIG. 10. In vitro bioassays demonstrating growth retardation of EMRSA-15with Sig complexes formed with Compound 7.

FIG. 11. In vitro bioassays demonstrating growth retardation of E. coliwith EC_Fur complexes formed with Compound 7.

FIG. 12. In vitro bioassays demonstrating growth retardation of EMRSA-15with Sig complexes formed with Compound 7_12.

FIG. 13. Graphs of time elapsed against optical density showing theeffect of a hairpin Sig TFD/Compound 7_12 complex vs a Scrambled complexand an Empty control on the growth of the MRSA strain.

FIG. 14. Graphs of time elapsed against optical density showing theeffect of a hairpin Sig TFD/Dequalinium complex vs a Scrambled complexand an Empty control on the growth of the MRSA strain.

FIG. 15. Graph illustrating the weight gain of mice treated with SA_SigTFD/Compound 7 complex, SA_Sig TFD/Compound 7_12 complex, or salinesolution. a) and b) denote independent repeats of the experiment.

FIG. 16. Bar chart showing kidney tissue burden following treatment ofmice with Compound 7_12, SA_Sig TFD/Compound 7_12 complex, SA_Sig_ScrTFD/Compound 7_12 complex, SA_Sig TFD, Vancomycin, or Vehicle.

FIG. 17. In vitro bioassays demonstrating growth retardation of EMRSA-15with Sig TFD mixed with Gramicidin.

FIG. 18. In vitro bioassays demonstrating growth retardation of EMRSA-15with Sig TFD mixed with Buforin II.

FIGS. 19A and 19B. Fluorescent microscopy confirms delivery ofdye-labelled oligonucleotide to MRSA by Buforin II-derivatised complexesformed with Compound 7.

FIG. 20. In vitro bioassays demonstrating growth retardation of E. coliwith EC_Fur TFD mixed with Polymyxin.

FIGS. 21A and 21B. Fluorescent microscopy confirms delivery ofdye-labelled oligonucleotide to MRSA by Buforin II-derivatised complexesformed with Compound 7.

FIG. 22. In vitro bioassays demonstrating growth retardation of EMRSA-15with Buforin II-derivatised Sig TFD complexes formed with compound 7.

BRIEF DESCRIPTION OF THE SEQUENCES

-   SEQ ID NO: 1—a native WhiB7 binding site in M. smegmatis str MC2 155-   SEQ ID NO: 2—WhiB7 transcription factor decoy-   SEQ ID NO: 3—a native FadR binding site in E. coli K12-   SEQ ID NO: 4—FadR transcription factor decoy-   SEQ ID NO: 5—a native binding site for YycF/YycG in S. aureus-   SEQ ID NO: 6—a native binding site for YycF/YycG in S. aureus-   SEQ ID NO: 7—LytM decoy-   SEQ ID NO: 8—SsaA decoy-   SEQ ID NO: 9—a native binding site for Sig^(B) in S. aureus-   SEQ ID NO: 10—a native binding site for Sig^(B) in K. pneumoniae-   SEQ ID NO: 11—a consensus sequence for Fur binding in S. aureus-   SEQ ID NO: 12—a consensus sequence for Fur binding in E. coli-   SEQ ID NO: 13—a native binding sequence for Fur in H. pylori-   SEQ ID NO: 14—a consensus binding site for TcdR in C. difficile-   SEQ ID NO: 15—a consensus binding site for Vfr in P. aeruginosa-   SEQ ID NO: 16—a native binding site for Vfr in P. aeruginosa-   SEQ ID NO: 17—a native binding site for Vfr in P. aeruginosa-   SEQ ID NO: 18—a native binding site for NtrC in K. pneumoniae-   SEQ ID NO: 19—a native binding sequence for ArsR in H. pylori-   SEQ ID NO: 20—a native binding sequence for ArsR in H. pylori-   SEQ ID NO: 21—a glycopeptide-resistant consensus sequence in S.    aureus-   SEQ ID NO: 22—an Agr binding motif in S. aureus-   SEQ ID NO: 23—an Agr binding motif in S. aureus-   SEQ ID NO: 24 & 25—forward and reverse primer sequences for PCR    preparation of the SAsigB TFD-   SEQ ID NO: 26 & 27—forward and reverse primer sequences for PCR    preparation of the SAfhu TFD-   SEQ ID NO: 28 & 29—forward and reverse primer sequences for PCR    preparation of the SsaA TFD-   SEQ ID NO: 30—phosphorylated Sig dumbbell TFD oligonucleotide    sequence-   SEQ ID NO: 31—phosphorylated Sig dumbbell TFD oligonucleotide    sequence-   SEQ ID NO: 32—phosphorylated oligonucleotide incorporating the    binding site for WalR-   SEQ ID NO: 33—phosphorylated oligonucleotide incorporating the    binding site for WalR-   SEQ ID NO: 34 & 35—forward and reverse primers for FabB promoter-   SEQ ID NO: 36 & 37—forward and reverse primers for TFD containing    the recognition sequence for the σ54 factor of K. pneumoniae-   SEQ ID NO: 38—WalR TFD consensus sequence-   SEQ ID NO: 39—SigB TFD consensus sequence-   SEQ ID NO: 40—KP_Sig TFD sequence-   SEQ ID NO: 41—KP_Sig TFD consensus sequence-   SEQ ID NO: 42—Gram negative Sig TFD hairpin sequence-   SEQ ID NO: 43 & 44—forward and reverse primers for scrambled S.    aureus Sig binding site-   SEQ ID NO: 45 & 46—forward and reverse primers used for    amplification of a target sequence from the pGEMT-Easy vector-   SEQ ID NO: 47 & 48—forward and reverse primers for WhiB7 TFD-   SEQ ID NO: 49—SA SIG hairpin TFD sequence-   SEQ ID NO: 50—SA_SIG scrambled hairpin TFD sequence-   SEQ ID NO: 51—Buforin II peptide sequence-   SEQ ID NO: 52—Tef-derivatised SASig TFD

EXAMPLE 1 Formation of Complexes with Dequalinium Analogues and TFDs

Weissig et al have shown (WO99/013096) that dequalinium (DQA) can beused to deliver DNA to mitochondria. With sonication, DQA formsspheric-appearing aggregates with a diameter of between about 70 and 700mm, which is similar to phospholipids vesicles. These aggregates weretermed ‘DQAsomes’ in WO99/013096 and ‘bolasomes’ in Weissig andTorchilin ((2001) Adv. Drug Delivery Rev. 49: 127-149). The term‘bolasome’ is used in this specification to describe vesicles of DQA andits analogues after the compounds have been subjected to sonication.

Complexes consist of a Transcription Factor Decoy (TFD) oligonucleotideself-assembled with a suitable delivery compound. A TFD oligonucleotideis 40 to 100 nucleotides in length and has a natural phosphate backbone.It self anneals to form a binding site for the targeted transcriptionfactor and has a naturally-forming hairpin to protect the 5′ and 3′ends, an example of which is shown below (GN_SIG_HP):

SEQ ID NO: 42 -agc-gtg-ata-atc-att-atc-g- agcg5′ g3′-cac-tat-tag-taa-tag-a-

In an alternative configuration, small hairpin loops at either end ofthe TFD act to protect the molecule from degradation and give the TFD adumbbell (DB) shape.

Materials and Methods.

Preparation of delivery compounds. 15 mg of each compound (SygnatureLtd.) was dissolved in 10 ml methanol and dried to completion using arotary evaporator and re-suspended in 5 mM Hepes pH7.4 to a finalconcentration of 10 mM. Compound 7 dissolved readily to give a clear,light yellow solution. Compound 7_12 dissolved after being place in asonicator bath for 1 h, forming an opaque, light yellow solution. Bothsolutions were subjected to probe sonication on ice using an MSESoniprep 150. The conditions used were: 60 cycles of 30 s on (amplitude10 microns) and 60 s off. Following this treatment, the Compound 7_12sample was entirely clear. Both samples were centrifuged to removedebris and are referred to as sC7 (sonicated Compound 7) or sC7_12(sonicated Compound 7_12). This step formed vesicles or ‘bolasomes’.

Preparation of Dumbbell TFD Complexes. 2 μg of TFD (a 32 bpoligonucleotide which has been ligated to form a monomeric circle) wasmixed with 1 ml of either 5 mM Hepes pH7.4 buffer or LB broth (LuriaBertani broth: 1% (w/v) Bacto-tryptone, 5% (w/v) Bacto-Yeast Extract, 5%(w/v) NaCl) which was then mixed with between 1 and 10 μl of either sC7or sC7_12 at room temperature.

Preparation of Hairpin TFD Complexes. Oligonucleotides were suspended inwater at a concentration of 1 mM (i.e. 180 nmoles in 180 μl). Thesuspension was diluted to 10 μM in water and heated to 95° C. for 2 minsin dry heating block, after which the suspension was removed from theheat and allowed to cool to room temperature. To confirm that the TFDhad annealed properly, 1 μl TFD was mixed with 1 μl 10×NEB Buffer 1, 6μl water and 1 μl Exonuclease I (NEB). A control mixture excludes the 1μl TFD. The mixture was incubated at 37° C. for 30 min before beingseparated on a 3% Low Melting Point agarose gel/TAE stained with0.5×SYBR Green. Correct TFD conformation was confirmed by resistance toexonuclease digestion.

To prepare delivery complexes, 10 mg of compound was suspend in 12.5 mlof 5 mM Hepes pH7.5 (final concentration 0.8 mg/ml) and dissolved bysonication (30×30 s on, 30 s off, on ice at 10μ). Absorbance of theresulting solution was measured at 327 nm to establish an accurateconcentration. 12.5 μl delivery compound was mixed with 40 μl 10 μM TFDand 447.5 μl 5 mM Hepes (pH7.5) and sonicated in an ice bath using anMSE 150 Soniprep attached with a microprobe. Thirty cycles of sonicationwere performed with 30 s on (with 50% power, approximately 10μ) and 30 soff.

DNA Binding Assays. To determine the proportion of TFDs being bound bythe vesicles, a SYBR-green binding assay was used. TFD complexes wereformed as described above in Hepes buffer with the adaptation that thebuffer contained 5 μl of a 1 in 10 dilution of SYBR Green I dye(Invitrogen, 10,000× stock prepared in DMSO). Fluorescence was measured(λ_(EX) 497 nm, λ_(EM) 520 nm) to determine how much bolasome needed tobe added to quench the binding of SYBR Green to the TFDs.

Size Determination. The size of the bolasomes was determined by DynamicLight Scattering using a Dynapro Titan DLS Instrument.

Visualisation of Particles. The size of the particles was measured usingelectron microscopy. Samples were directly stained with uranyl acetatebefore imaging.

Results.

1.1 Size Distribution of sC7 Bolasomes

sC7 bolasomes were prepared in 5 mM Hepes buffer at a concentration of10 mM. Prior to measurement of their size distribution by dynamic lightscattering the bolasomes were diluted in the same buffer 1000-fold. Themajority of the material by mass had a diameter in excess of 3 μm andwas caused by non-specific aggregates or dust. The remaining particleshad an average diameter of 68 nm (FIG. 4). This is somewhat different tothe published size distribution of the vesicles (Weissig et al. 2001 S.T. P. Pharma Sci. 11: 91-96: Table I, see Compound 7) which estimatedthe size distribution to be 169 nm+/−50 nm and commented that thedistribution was tight. The values for sC7 bolasomes are closer to thepublished values, although the difference may reflect differentexperimental parameters and measurement instruments. Indeed, the factthat the sC7 bolasomes were stable despite dilution indicates that theyhave increased stability over bolasomes formed by sonication ofdequalinium solutions, as these revert to the monomer on dilution (whichhas a diameter of less than 10 nm).

The diameters of sC7 and sC7_12 bolasomes (see 1.2) are tabulated inTable 2:

TABLE 2 Calculated values of minimum concentrations of sC7 and sC7_12bolasomes needed to quench SYBR Green-binding to a fixed concentrationof TFD. Item Diameter (nm) % Mass sC7 bolasomes Peak 1 68.0 100 sC7_12bolasomes Peak 1 48.4 27.2 Peak 2 197.7 72.81.2 Size Distribution of sC7_12 Bolasomes

sC7_12 bolasomes were prepared in 5 mM Hepes buffer at a concentrationof 10 mM. Prior to measurement of their size distribution by dynamiclight scattering the bolasomes were diluted in the same buffer1000-fold. As described in 1.1, the signal from the large material wasdiscounted. The remaining particles had average diameters of either 48.4nm or 197.7 nm and were present in a ratio of 1:2.5 (FIG. 5). This wasmarkedly different from the diameters of the sC7 bolasomes. However, theparticles had a better size distribution than reported by others forthose formed with similar concentrations of dequalinium and werecomparable to those obtained by Weissig for bolasmomes formed fromCompound 7 (Weissig et al. (2001) S. T. P. Pharma Sci. 11: 91-96).

1.3 Establishing Optimum Binding Conditions of sC7 and sC7_12 to TFDwith DNA-binding Assay

SYBR-Green I dye binds specifically to double-stranded DNA and, as itdoes, gives a strong fluorescent signal. By measuring the change insignal in the presence of different concentrations and types ofbolasome, it was possible to calculate the minimum amount of bolasomeneeded to quench the SYBR-Green binding, due to the dye being excludedby the bolasomes. This was achieved by extrapolation from the linearportion of a titration curve that plotted amount of bolasome added tofluorescent signal. Using a fixed concentration of 2 g TFD/ml, theminimum concentration of sC7 bolasome was found to be 6.13 μg/ml (FIG.6). At these concentrations no quenching was seen by the monomeric C7.

The minimum concentration for the sC7_12 bolasomes was found to be 9.26μg/ml (FIG. 7).

The minimum amount of bolasome required was used in the preparation ofTFD complexes. Such concentration was also used to ensure that there wasas little sample to sample variation as possible between thepreparations of bolasomes. In general, variation of approximately 20%was seen and had no observable affect on biological function.

The stability of the complexes was measured by monitoring the normalisedfluorescence due to SYBR Green dye binding. The titration curves for TFDcomplexes formed with sC7 bolasomes remained constant for excess of 72 hwhen stored at 4° C. (FIG. 8), showing that the conditions illustratedhere provide a substantial improvement in the stability of the complexesformed.

1.4 Electron Micrograph Imaging of sC7 Bolasomes and TFD Complexes

The TFD complexes formed between the sC7 bolasome and a TFD werevisualised by electron microscopy by negative staining with uranylacetate. Two examples are shown in FIGS. 9A and 9B. Round particles ofbetween 50 and 100 nm were clearly seen with densely staining interiorswith granules evident in the interior that may be condensed bodies ofDNA.

Example 2 Delivery of TFD in Compound 7 Bolasome Kills MRSA

Materials and Methods

Preparation of TFD Dumbbells by Ligation (DB-TFD)

Two oligonucleotides were synthesised, each containing one strand of therecognition site for the S. aureus alternative sigma protein. At eitherend of the molecule a small hairpin loop acted to protect the moleculefrom degradation. Each oligonucleotide was re-suspended in dH₂O at aconcentration of 250 pmol/μl. To form the Sig dumbbell TFD (referred toas Sig TFD) the following phosphorylated oligonucleotides weresynthesised:

SigDB1: SEQ ID NO: 30 CTT GGT TTT TCC AAG GAA GAT TAG AAA TTA TTT CGATGG GTA TAT AAT A SigDB2: SEQ ID NO: 31 P-CCG TCT TTT TGA CGG TAT TATATA CCC ATC GAA ATA ATT TCT AAT CTT C

When annealed, these formed the following molecule:

T CCAAG gaa gat tag aaa tta ttt cgat ggg tat ata ata PCCGTC T TTT TTT TGGTTCPCTT CTA ATC TTT AAT AAA GCTA CCC ATA TAT TAT GGCAG T

30 μl of each oligonucleotide was mixed with 27 μl of dH₂O and annealedusing the following PCR programme: ANNEAL: 95° C. 3 min, cool at −0.1°C./s to 8° C., end. Following which, 10 μl of 10×NEB Ligase buffer and 3μl HC T4 DNA ligase (NEB) were added. The mixture was incubatedovernight at 16° C. The material was then extensively digested with T7exonuclease (NEB) to remove any unligated oligonucleotides and thenrecovered by two rounds of ethanol precipitation. A DB_TFD was alsoprepared containing a scrambled version of the Sig binding site,referred to as Scr TFD. In this instance the phosphorylated primers usedwere:

SigScr_SA1: SEQ ID NO: 43 CTT GGT TTT TCC AAG TAG AAA GAA GAT TTA GGGCGA T TTT ATA ATA TAT SigScr_SA2: SEQ ID NO: 44 CCG TCT TTT TGA CGG ATATAT TAT AAA ATC GCC CTA AAT CTT CTT TCT AFormation of Complexes

The minimum amount of sC7 bolasome needed to bind 2 μg of either TFD wasestablished empirically and the appropriate amount of bolasome was mixedwith the TFDs in 5 mM Hepes, pH7.4, to form complexes. Dilutions of theTFD nanoparticle were used in subsequent bioassays.

Performing Growth Studies in 96-well Plates

A growth assay was performed using complexes consisting of either theSig TFD or Scr TFD mixed with sC7 bolasomes, to determine the effect ongrowth of a clinically-isolated MRSA strain. The assays to determine theeffect on growth of bacterial cells were performed using 96 well plates,each well containing 200 μl of broth consisting of LB media. 1 μl ofvarious concentrations of TFD complexes was added to each well and theeffect on bacterial growth of S. aureus was monitored by measuringabsorbance of the broth at intervals during incubation. The plates wereincubated at 37° C. with shaking and absorbance readings (at 450 nM)were taken using a plate reader.

Results

2.1 TFD Complexes can Efficiently Kill MRSA In Vitro

TFD complexes were prepared with sC7 bolasomes using a TFD known to killMRSA cells called ‘Sig TFD’ or a scrambled version as a control, ‘ScrTFD’. The MRSA strain, EMRSA15, was used to inoculate LB broth toprovide a final concentration of cells of 5×10⁵/ml. 200 μl aliquots weredispensed into wells in a 96 well plate. Wells were supplemented withvarying concentrations of Sig TFD complex, Scr TFD complex, equivalentconcentrations of the sC7 bolasomes as a control for any antibacterialeffect of the dequalinium analogue (sC7 control) or the wells wereuntreated (FIG. 10). Both TFD complexes contained sC7 at a concentrationof 500 ng/ml and TFDs at 5 μg/ml. The sC7 control consisted of bolasomesat a concentration of 500 ng/ml.

Cell growth was essentially similar for the untreated sample, the sC7control and the Control Complex (consisting of the Scr TFD). However,the Sig TFD complex prevented bacterial growth. Hence, the combinationof the sC7 bolasome with the Sig TFD killed the MRSA strain, whereas thecontrol TFD complex did not. This was due to the complexes effectivelydelivering the TFD therapeutic to the MRSA. The action of deliveryalone, with concomitant membrane damage, did not kill the bacteria asneither the Control Complex nor an equivalent amount of sC7 bolasomesaffected cell growth.

Example 3 Delivery of TFD by Compound 7 Bolasome Kills E. coli

Materials and Methods

Preparation of Fur TFDs by PCR

Fur TFDs were designed to incorporate the binding site for thetranscriptional regulator of fatty acid synthesis enzymes, FadR, whichoccurs upstream of the FabB gene in Escherichia coli. The FabB geneencodes an enzyme involved in fatty acid synthesis (J. Bacteriology(2005) 183:5292). The oligonucleotides used to amplify the promotersequence were:

fabBf SEQ ID NO: 34 5′-tct tta aat ggc tga tcg gac ttg-3′ fabBr SEQ IDNO: 35 5′-agt aag ttt cga atg cac aat agc gta-3′

The resulting fragment was ligated into pGEMTEasy vector (Promega) andPCR TFDs were synthesized by PCR amplification using oligonucleotideprimers designed to anneal to the backbone of the vector immediatelyflanking the insert, for example:

TEf: SEQ ID NO: 45 5′-ggc cgc cat ggc ggc cgc ggg aat tc-3′ TEr: SEQ IDNO: 46 5′-agg cgg ccg cga att cac tag tg-3′.

The PCR product is ethanol precipitated and re-suspended in TE buffer(10 mM Tris.HCl, 1 mM EDTA pH8.0) at a concentration of 500-1000 ng/μl.

A control TFD having a the sequence that gave rise to a similar sizedPCR fragment when used in an amplification reaction with genomic DNAisolated from Mycobacterium smegmatis was also generated. The sequencesof these oligonucleotides were:

WhiB7.f SEQ ID NO: 47 CAC CAG CCG AAA AGG CCA CGG WhiB7.r SEQ ID NO: 48CAA AAA TGG CCA CGG ATC CGG GTGResults3.1 TFD Complexes can Efficiently Kill E. coli In Vitro

TFD complexes were formed with a TFD known to be active against E. coli,EC Fur, and a control TFD, EC FurScr TFD. The experiment was performedas described in Example 2.1 and similar results were obtained (FIG. 11).Again, the results show that complexes formed with sC7 bolasomes and ECFur TFD prevented growth of E. coli (strain DH10B) in an iron-limitedmedia.

Example 4 Delivery of TFD by Compound 7_12 Bolasome Kills MRSA

Materials and Methods

TFD complexes were formed as described in Example 2 with Sig TFD or ScrTFD, with the exception that sC7_12 bolasomes were used. The resultantTFD complexes were tested for their activity in preventing growth ofMRSA strain EMRSA15.

Results

4.1 TFD Complexes can Efficiently Kill MRSA In Vitro

TFD complexes were prepared using a TFD known to kill MRSA cells called‘Sig TFD’ and a scrambled version, ‘Scr TFD’, as a control with sC7_12bolasomes. The MRSA strain, EMRSA15, was used to inoculate LB broth togive a final concentration of cells of 5×10⁵/ml. 200 μl aliquots weredispensed into wells in a 96 well plate. Wells were supplemented withvarying concentrations of Sig TFD complexes, Scr TFD complexes,equivalent concentrations of the sC7_12 bolasomes as a control for anyantibacterial effect of the dequalinium analogue (sC7_12 control) or thewells were untreated (FIG. 12). Both TFD complexes contained sC7_12 at aconcentration of 800 ng/ml (1.3 μM) and TFDs at 5 μg/ml (153 nM). ThesC7_12 control consisted of bolasomes at a concentration of 800 ng/ml.

Cell growth was essentially similar for the untreated sample, the sC7_12control and the control complex (including the Scr TFD). However, theSig TFD complex prevented bacterial growth. Hence, the combination ofthe sC7_12 bolasome with the Sig TFD killed the MRSA strain, whereas thecontrol TFD complex did not. This was due to the complexes effectivelydelivering the TFD therapeutic to the MRSA. The action of deliveryalone, with concomitant membrane damage, did not kill the cells asneither the control complex nor an equivalent amount of sC7_12 bolasomesaffected cell growth.

Example 5 Delivery of Hairpin TFD by Various Delivery Compounds KillsMRSA

TFD complexes containing the hairpin TFD SA SIG and either dequalinium,Compound 7 or Compound 7_12 were prepared using the method set out inExample 1. The size distributions of the formed vesicles were measuredusing a Malvern Nanosizer using standard methodology and are set out inTable 3 below:

TABLE 3 Delivery Compound Vesicle size distribution (nm) Concentration(μM) Dequalinium 75-820 745 Compound 7 139 +/− 35 75 Compound 7_12 117+/− 32 75

The size distribution of the vesicles was found not to alter when TFDsof different sequences were used. The sequence of the SA SIG HP(targeted to bind to the alternative sigma factor in Staphylococcusaureus) is:

SEQ ID NO: 49 5′-gcg aag cga aga tta gaa att att tcc atg ggt ata taa tacttg gtt ttt cca agt att ata tac cca tgg aaa taa ttt cta atc ttc-3′5.1. Efficacy of SA_SIG_HP TFD Complexed with Compound 7_12

The TFD complex was prepared as described in Example 1 (referred to asSig) as were two control snares, one containing no TFD (Empty) and theother a scrambled version of SA SIG HP (Scrambled) that contained theTFD SA_SIG_Scr_HP, which has the following sequence:

SEQ ID NO: 50 5′-gcg aag cat ctt gta tgc aaa tag aat gaa taa tag ttt gacttg gtt ttt cca agt caa act att att cat tct att tgc ata caa gat-3′

1 μl of each delivery complex was added to 200 μl of LB broth inoculatedwith 1 μl of a glycerol stock of an MRSA strain, EMSRA15, at aconcentration of 3×10⁶ colony forming units per μl. The cultures weregrown at 37° C. with mild shaking and the optical density of thecultures measured in a plate reader at half hour intervals.

The plots of time elapsed against optical density shown in FIG. 13demonstrate that the Sig complex effectively prevented growth of theMRSA strain with concentrations of 500 ng/ml Compound 7_12 and 20 pmolTFD. Bacteria treated with the Scr complex (with similar concentrationsof both Compound 7_12 and TFD) grew slower than both Empty control andthe untreated sample. This has been observed in previous experiments andis interpreted as being due to growth being slowed by the action ofdelivering the scrambled TFD into the cell. When that TFD inhibitsstress response, as does SA_SIG_HP, the cells fail to recover. Growth ofthe Empty control and the untreated sample were indistinguishable.

Hence, complexes containing the SIG_SA_HP TFD, designed to blockessential pathways in S. aureus, are fatal to bacterial cells whereasdelivery of a scrambled version of the TFD or the delivery vehicle aloneis ineffective.

5.2. Efficacy of SA_SIG_HP Complexed with Dequalinium

The TFD complex was prepared as described in the section 5.1 above withthe exception that the concentration of dequalinium used was 6-foldhigher than the concentration of Compound 7_12. Similarly two controlsnares were prepared, one containing no TFD (Empty) and the other ascrambled version of SA SIG HP (Scrambled) that contained the TFDSA_SIG_Scr_HP.

1 μl of each delivery complex was added to 200 μl of LB broth inoculatedwith 1 μl of a glycerol stock of an MRSA strain, EMSRA15 at aconcentration of 3×10⁶ colony forming units per μl. The cultures weregrown at 37° C. with mild shaking and the optical density of thecultures measured in a plate reader at half hour intervals. The plots oftime elapsed against optical density shown in FIG. 14 demonstrate thatthe Sig antibacterial effectively prevented growth of the MRSA strainwith concentrations of 3 μg/ml Compound 7_12 and 10 pmol TFD. Bacteriatreated with the Scr antibacterial (with similar concentrations of bothDequalinium and TFD) grew slower than both Empty control and theuntreated sample. This has been observed in previous experiments and isinterpreted as being due to growth being slowed by the action ofdelivering the scrambled TFD into the cell. When that TFD inhibitsstress response, as does SA_SIG_HP, the cells fail to recover. Growth ofthe Empty control and the untreated sample were indistinguishable.

Hence, complexes containing the SIG_SA_HP TFD, designed to blockessential pathways in S. aureus, is fatal to the cells whereas deliveryof a scrambled version of the TFD or the delivery vehicle alone areineffective.

Example 6 Efficacy of SA Sig TFD by Compound C₇₋₁₂ in Treatment of MRSAin a Mouse Sepsis Model

Mice used in this study, male CD1 mice, were supplied by Charles River(Margate UK) and were specific pathogen free (16-18 g at delivery). Allmice weighed 22-25 g at the beginning of the experiment.

6.1. Tolerability Study

Animals were treated in groups of two mice per treatment group,therefore six animals were used in total for the study. All the micewere weighed on day 1 of the study and placed randomly into boxes. Themice had the following treatments administered intravenously at 10ml/kg:

-   -   100 μM SA Sig TFD (2 mg/ml; SEQ ID NO:9) and 525 μM (0.315        mg/ml) Compound 7 in saline solution;    -   100 μM SA Sig TFD (2 mg/ml; SEQ ID NO:9) and 525 μM Compound        7_12 (0.315 mg/ml) in saline solution; and    -   saline solution alone.

The concentrations of TFD and delivery molecules were chosen to beapproximately ten-fold greater than the predicted effective dose.

The mice were weighed daily post-treatment over a 100 h period beforethey were euthanised. The lungs, liver, spleen and kidneys were removedand visually examined and weighed. FIG. 15 shows the weight gain of allthree groups with indices a) and b) referring to independentexperimental repeats.

No significant difference was seen between the control treatments(saline) and those treated with combinations of TFD and deliverycompounds. Thus, all treatments were well tolerated followingintravenous administration. There were no acute events to report.Following treatment, mice fed and drank normally with no signs ofdistress. The weight increase of the treated mice was the same as thevehicle controls. Autopsy showed no gross abnormalities of kidneys,lungs, liver or GI tract. The weights of kidneys, lungs and liver werewithin the normal range. All test compounds are tolerated and suitablefor further dosing up to the maximum dose used in this tolerabilitystudy.

6.2. Tissue Burden Study

Animals were treated in groups of since mice per treatment group,therefore forty eight animals were used in total for the study. Two 10ml cultures of Staphylococcus aureus EMRSA 16 were prepared and placedon orbital shaker (220 rpm) overnight at 37° C. The following day, theStaphylococcus aureus EMRSA 16 cultures were removed from shaker,pelleted and washed twice before being resuspended in saline to an OD of0.132 (1.5×10⁸ cfu/ml). This stock solution of Staphylococcus aureusEMRSA 16 was then further diluted 1:1.5 in saline (1×10⁸ cfu/ml) i.e.2.0×10⁷ bacteria per mouse.

All forty eight mice were then infected with 0.2 ml of the 1.0×10⁸/mlsuspension by intravenous injection into mouse tail vein. The number ofStaphylococcus aureus EMRSA 16 bacteria per ml in the remainder of thesuspensions after inoculation was also counted to confirm infectionload.

Mice were treated 1, 9 and 17 hours post infection with either compoundor vehicle, though vancomycin was only administered after 1 h. Thetreatments were a combination of antibiotic complexes, prepared withCompound 7_12 and various TFDs, as tabulated in Table 3 below.

TABLE 3 Compound SA Scr Sig Treatment C7_12 SA Sig TFD TFD VancomycinC7_12 alone 15 ng/kg — — — Sig complex 15 ng/kg 1 nM — — Scr complex 15ng/kg — 1 nM — Sig TFD — 1 nM — — Vancomycin — — — 25 mg/kg Saline — — ——

After 25 hours post infection, all animals were weighed and theneuthanised. The kidneys were immediately removed and homogenised inice-cold sterile phosphate buffered saline+0.05% Tween 80. Organhomogenates were quantitatively cultured onto CLED agar and incubated at37° C. for up to 3 days and colonies counted. The data from the cultureburdens was analysed by the Kruskal-Wallis test using Stats Direct.

6.3. Tissue Burden Study

The infectious dose administered was targeted at 3.7×10⁷ bacteria permouse to ensure that a relatively acute infection was established i.e.an infection that is sensitive to treatment. The mice were treated withsystemic injection of either (A) the delivery compound alone (CompoundC7_12), (B) 1 nM of SA_Sig/Compound 7_12 complex, (C) 1 nM of Scrambledcontrol complex, (D) 1 nM of SA_Sig TFD alone, (E) vancomycin, used at aconcentration sufficient to achieve a 2-fold reduction in colony formingunits (cfu), or (F) vehicle. Following treatment, the mice weresacrificed and the burden found within the kidneys measured (see FIG.16).

Statistical analysis of the results showed that the Sig snareantibacterial-treated mice achieved a similar reduction in burden tothat achieved by vancomycin. All other controls show similar burdens tothe vehicle treatment (Table 4).

TABLE 4 Statistical analysis of in vivo results. Kruskal-Wallis: allpairwise comparisons (Dwass-Steel-Chritchlow-Fligner) for Sig TFDcomplex Sig Scr Sig Treatment complex complex TFD Vancomycin VehicleC7_12 0.0001 0.5490 0.4695 0.0004 0.4109 Sig snare 0.0008 <0.0001 0.7263<0.0001 Scr snare 0.1894 0.0023 0.1588 Sig TFD <0.0001 0.9203 Vancomycin<0.0001

The Sig complex in this experiment was found to have a rapidbacteriocidal activity at nanomolar concentrations against MRSA both invitro and in vivo.

Example 7 Delivery of TFDs Mediated by Antibacterial Peptides

Materials and Methods

The following antibacterial peptides were assayed for their ability todeliver TFDs to bacterial cells: Gramicidin, Polymyxin nonapeptide (bothpurchased from Sigma Aldrich) and Buforin II (Park et al. (2000) Proc.Natl. Acad. Sci. USA 97: 8245-8250). Typically 1 μg of the Sig DB-TFDsand SigScr DB-TFD as described in Example 1 were mixed with between 0.2and 5 μg of Gramicidin in a total volume of 5 μl 50 mM NaCl. Of this, 1μl was added to 200 μl of LB broth inoculated with a 1/100 dilution of aglycerol stock of EMRSA-15 at an original density of 0.3 OD (Absorbanceat 600 nm) and aliquoted into a well of a 96 well plate. Experimentswere performed in triplicate. The plates were incubated at 37° C. withshaking and absorbance readings (at 450 nM) were taken using a platereader.

Results

7.1. Gramicidin Effectively Delivers TFDs to S. aureus

Adding 1 μg of Sig DB-TFD to LB media inoculated with EMRSA-15 and with150 ng/ul of Gramicidin resulted in no bacterial growth. In contrast theTFD alone, Gramicidin alone or Gramicidin mixed with the scrambledversion of the DB-TFD grew as well as the untreated control (FIG. 17).

7.2. Buforin II Effectively Delivers TFDs to S. aureus

The 21 amino acid Buforin II peptide consisted of the followingsequence: TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO:51). Sig TFDs mixed withBuforin II retarded growth of EMRSA-15 when mixed with themembrane-active antimicrobial peptide Buforin II and prevented growth ofthe bacteria in a 96 well plate in vitro assay. The control TFD, Scr,which was a scrambled version of the sequence in the Sig TFD, had nodiscernable effect on growth when compared to the untreated broth or thecells treated with peptide alone (FIG. 18).

As an alternative, a fluorescently-labelled oligonucleotide was used asa substitute for the TFD. Though this has no predicted activity as a TFDmolecule, the fluorescein-labelled oligonucleotide incorporates afluorescent label so its uptake can be monitored by fluorescent lightmicroscopy (FIG. 19).

7.3. Polymyxin Effectively Delivers TFDs to E. coli

When mixed with the Gram-negative active antimicrobial cyclicglycopeptide polymyxin, EC Fur TFD retarded growth of DH5a and preventedgrowth of the bacteria in a 96 well plate in vitro assay usingiron-limiting media. The control TFD, WhiB7 TFD which was an unrelatedsequence of similar size as the Fur TFD, had no discernable effect ongrowth when compared to the untreated broth or the cells treated withpeptide alone (FIG. 20).

Example 8 Delivery of TFD/Dequalinium Complexes Conjugated with BuforinII

Materials and Methods

Derivatisation of Complexes with Antimicrobial Peptide

TFD complexes were derivatised with the antimicrobial peptide Buforin IIusing the cross-linker EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; ThermoScientific) and following an adaptation of the two-step couplingprotocol as described by the manufacturer.

The Buforin II peptide was synthesised and lyophilised and re-suspendedat a concentration of 1 mg/ml in Activation Buffer (0.1 M MES[2-(N′-morpholino)ethane sulfonic acid], 0.5 M NaCl, pH6.0). 1 ml ofthis solution was mixed with 0.4 mg EDC and 1.1 mg Sulfo-NHS (ThermoScientific) and incubated for between 5 and 120 min at room temperature,most typically 30 min. After incubation, 1.4 μl 2-mercaptoethanol wasadded to quench the EDC. Unincorporated EDC and Sulfo-NHS were removedfrom the peptide sample by dialysis using tubing with a low molecularweight cut-off (Pierce, Slide-A-Lyzer, 2K MWCO).

Concentrated TFD complexes were prepared using techniques described inExamples 2 and 3. 35 μl of TFD at a concentration of 1.5 mg/ml was mixedwith 130 μl PBS (Phosphate-buffered saline; 0.1 M sodium phosphatebuffer pH7.2, 0.15 M NaCl) and 35 μl of sonicated Compound 7 (bolasomes)at a concentration of 3.15 mg/ml. Typically 90 μl of concentrated TFDcomplexes were mixed with 10 μl of derivatised Buforin II and allowed toreact for 2 hours at room temperature. The reaction was quenched byaddition of 10 mM hydroxylamine. Prior to use in bioassays thederivatised TFD nanoparticle were diluted to the appropriateconcentration.

As an alternative, a fluorescein-labelled oligonucleotide was used as asubstitute for the TFD. Though this has no predicted activity as a TFDmolecule, the fluorescein-labelled oligonucleotide incorporates afluorescent label so its uptake can be monitored by fluorescent lightmicroscopy.

Results

8.1. Buforin II-derivatised TFD Complexes Effectively DeliverOligonucleotides to S. aureus

Derivatised TFD complexes were formed as described, with the exceptionthat the TFD was substituted with a fluorescently labelledoligonucleotide, Tef, that contained a fluorescein dye at the 5′ end.The sequence of the oligonucleotide was:

Tef-Fluorescein- SEQ ID NO: 52 AGG CGG CCG CGA ATT CAC TAG TGA.

The derivatised complexes are added to 200 μl of LB broth inoculatedwith the MRSA strain, EMRSA-15, and grown overnight with shaking at 37°C. The following morning, cells were harvested by centrifugation andwashed four times in an equal volume of PBS. A drop of the bacterialsuspension was placed on a microscope slide and air dried. Cells werethen heat-fixed by passing the slide through a Bunsen flame. The slidewas then flooded with a solution of Loeffler's methylene blue (5 mg/mlmethylene blue in 69:30:1 (v/v) solution of water:methanol:1% (w/v) KOH)and allowed to stand for 1 min, after which the excess solution waswashed off with water and the cells visualised using a Cairn CCDFluorescence Microscope.

In the bright-field view (no fluorescence) the bacteria could be clearlyseen clumped together (FIG. 21) and in the fluorescence view it could beseen that the labelled oligonucleotide had been internalised, consistentwith the derivatised nanoparticle affecting delivery. Bacteria grown inbroth without derivatised complexes showed no fluorescence.

8.2. Buforin II-derivatised TFD Complexes can Prevent Bacterial Growthof MRSA

Derivatised complexes were produced that contained either the Sig TFD orScr TFD as a control (as in Example 2). The concentration of the TFDcomponent in the stock of derivatised complexes was 8 μM and thecomplexes were diluted to give a working concentration of 16 nM TFD and1.8 μM Compound 7.

At this concentration the derivatised complexes containing the Sig TFDentirely prevented growth of the MRSA strain, while the derivatisedcomplex containing the control TFD did not. Indeed, growth was similarto the untreated sample and the sC7 control broth containing 1.8 μMCompound 7 bolasomes (FIG. 22).

Hence, complexes derivatised with Buforin II deliver Sig TFDs topathogenic bacteria to prevent growth. Furthermore, the effectiveconcentration of the complexes used was lower than that for thenon-derivatised complexes (section 4.1).

Example 9

Formation of Complexes with Other Dequalinium Analogues

By following the general synthetic methods described herein, thefollowing dequalinium analogues were prepared:

-   10,10′-(octane-1,8-diyl) bis (9-amino-1,2,3,4-tetrahydroacridinium)    diiodide;-   10,10′-(dodecane-1,12-diyl) bis    (9-amino-1,2,3,4-tetrahydroacridinium) diiodide;-   10,10′-(tetradecane-1,14-diyl) bis    (9-amino-1,2,3,4-tetrahydroacridinium) diiodide;-   10,10′-(octadecane-1,18-diyl)    bis(9-amino-1,2,3,4-tetrahydroacridinium) diiodide;-   5,5′-(dodecane-1,12-diyl)    bis(11-amino-7,8,9,10-tetrahydro-6H-cyclohepta[b]quinolinium)    diiodide;-   1,1′-(decane-1,10-diyl) bis (4-aminoquinolinium) diiodide;-   1,1′-(dodecane-1,12-diyl) bis (4-aminoquinolinium) diiodide;-   1,1′-(decane-1,10-diyl) bis (4-methoxyquinolinium) diiodide; and-   1,1′-(decane-1,10-diyl) bis (2-aminoquinolinium) diiodide.

The compounds listed above may be used in the methods described inExamples 1 to 8 to prepare further complexes according to the invention

What is claimed is:
 1. An antibacterial complex comprising: a doublestranded nucleic acid sequence comprising the sequence of a nativecellular binding site for a bacterial transcription factor; and one ormore delivery moieties represented by the formula:

wherein: A is a bond; p and q are the same or different and each is aninteger from 1 to 12; provided that the sum of p and q is in the rangefrom 8 to 18; R⁸ and R^(8a) are each selected from hydrogen; C₁₋₄alkoxy; nitro; amino; mono- and di-C₁₋₄alkylamino; and guanidinyl; R⁹ ishydrogen; R^(9a) is hydrogen; R¹⁰ is selected from hydrogen; amino; andC₁₋₄ alkyl optionally substituted with one or more fluorine atoms;R^(10a) is selected from hydrogen; amino; and C₁₋₄ alkyl optionallysubstituted with one or more fluorine atoms; or R⁹ and R¹⁰ link togetherto form an alkylene chain (CH₂)_(w) wherein w is 3 to 5: and/or R^(9a)and R^(10a) link together to form an alkylene chain (CH₂)_(w) wherein wis 3 to 5 provided that the compound of the formula is other thandequalinium, wherein the nucleic acid sequence is complexed with the oneor more delivery moieties.
 2. The antibacterial complex of claim 1,wherein the native cellular binding site comprises the sequence of abacterial SigB binding site.
 3. The antibacterial complex according toclaim 2, wherein the bacterial SigB binding site is represented by SEQID NOS: 9, 10, 30 and 31, 39, 40 or
 41. 4. The antibacterial complex ofclaim 1, wherein the native cellular binding site comprises the sequenceof a bacterial Fur binding site.
 5. The antibacterial complex of claim4, wherein the bacterial Fur binding site is represented by SEQ ID NOS:11, 12 or
 13. 6. The antibacterial complex of claim 1, wherein thebacterial infection is methicillin resistant.
 7. The antibacterialcomplex of claim 1, wherein the bacterial infection causes sepsis. 8.The antibacterial complex of claim 7, wherein the native cellularbinding site comprises SEQ ID NO:
 9. 9. The antibacterial complex ofclaim 1, wherein the alkyl chain has 12 or 14 methyl groups.
 10. Theantibacterial complex of claim 9, wherein the delivery moiety has theformula: